CN120958034A

CN120958034A - Methods and compositions for producing and purifying peptides

Info

Publication number: CN120958034A
Application number: CN202480014765.2A
Authority: CN
Inventors: O·苏布里尼; R·卡蒂尔; M·S·考博特
Original assignee: Micropeptide Technologies Ltd
Current assignee: Micropeptide Technologies Ltd
Priority date: 2023-02-23
Filing date: 2024-02-23
Publication date: 2025-11-14
Also published as: WO2024175778A2; CN120882737A; AU2024225703A1; IL322882A; WO2024175788A3; IL322884A; WO2024175780A1; AU2024226512A1; WO2024175778A3; KR20250154428A; WO2024175788A2

Abstract

Provided herein are methods for preparing peptides from inclusion bodies by ligating two or more oligopeptides to an insoluble carrier polypeptide (e.g., ranpirnase or TAF 12), wherein the oligopeptides are released from the insoluble carrier protein by sequence-specific chemical cleavage of peptide bonds using an acid such as acetic acid. Also provided herein are ranpirnase variants having improved properties.

Description

Methods and compositions for producing and purifying peptides

Reference to an electronic sequence Listing

The contents of the electronic sequence listing (185952001040 seqlist. Xml; size: 3,250,395 bytes; and date of creation: 2024, month 2, 7) are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to methods for expressing and purifying peptides from inclusion bodies.

Cross Reference to Related Applications

The present application claims the benefit of U.S. provisional patent application Ser. Nos. 63/486,652, 63/486,653, and 63/486,654, filed 2/23 of 2023, the entire contents of which are incorporated herein by reference.

Background

Although short peptides can be produced in high yields by chemical synthesis (Merrifield, R.B. (1993) J. Am. chem. Soc.85:2149-2154), recombinant production offers the potential for large-scale production at more reasonable cost. However, the biological production of short peptides presents special challenges. Peptides shorter than 30 amino acids in length are generally unstructured and therefore difficult to express individually in microbial culture by recombinant DNA techniques. Because of the potential toxicity of the short peptide to the host cell or the high sensitivity to proteolysis in vivo, which results in low yields of peptide if produced, the production of the short peptide by recombinant means is more complex (Gottesman, S. (1990) Methods in Enzymology 185:119-129; Goff and Goldberg, (1986) in Maximizing Gene Expression p287-314). although fusion of the short peptide to a larger carrier protein may help reduce toxicity, provide stability and facilitate purification (Terpe, k. (2003) appl. Microbiol. Biotechnol. 60:523-533), even when the peptide is part of a larger fusion protein, high concentrations of the fusion peptide in the cell may result in toxic effects.

The use of insoluble carrier proteins as fusion partners can help alleviate peptide toxicity and proteolytic degradation by facilitating aggregation of the peptide into insoluble inclusion bodies. Inclusion bodies prevent proteolysis and serve as purification substrates (t. Kempe et al., (1985) Gene 39:239-245). However, the usual methods for isolating peptides from carrier proteins may be inefficient and generally require expensive lysis reagents and affinity columns for purification. For example, protease-based recovery of peptides from carrier proteins using site-specific protease or autoprotease (autoprotease) fusion partners has been used with limited success, but these strategies require large peptide fusions and are incompatible with concatemeric peptide production strategies.

Peptide production methods using ranpirnase (onnase) as an insoluble carrier protein have been developed and provide a potential strategy for peptide production in bacteria or yeast. A recent ranpirnase-based strategy is described in Pane, K., et al (2016) PLoS one.11 (1): e0146552 ("Pane et al"). Pane et al expressed and purified peptides from inclusion bodies in E.coli using the ranpirnase-peptide fusion construct. The separation of peptides from ranpirnase is performed using a chemical cleavage strategy, which can be performed using low cost reagents, and generally targets peptide bonds that are present in proteins less frequently. Notably, although the protocol described by Pane et al requires the use of a chaotropic agent, the use of a chromatographic step is avoided. This approach still results in lower peptide yields and purities, which can potentially jeopardize downstream applications of the peptides produced by the method.

Thus, there remains a need for short peptide-producing methods that avoid toxic and proteolytic degradation, as well as allow simple, inexpensive and efficient recovery of peptides.

Disclosure of Invention

Aspect 1: a method of producing an oligopeptide, comprising:

Expressing a fusion polypeptide comprising an insoluble carrier polypeptide (insoluble carrier polypeptide) operably linked to 2 or more oligopeptides via peptide bonds, and

Releasing the 2 or more oligopeptides from the insoluble carrier polypeptide by sequence specific chemical cleavage of the peptide bond.

Aspect 2 the method of aspect 1, wherein the insoluble carrier polypeptide comprises a ranpirnase polypeptide.

Aspect 3 the method of aspect 1, wherein the insoluble carrier polypeptide comprises a TAF12 polypeptide.

Aspect 4 the method of aspect 1, wherein the insoluble carrier polypeptide comprises a trpΔLE polypeptide, a ketosteroid isomerase (KSI) polypeptide, a β -galactosidase polypeptide, a PagP polypeptide, a truncated E.coli PurF F fragment polypeptide, a P.aeruginosa (Pseudomonas aeruginosa) PaP3.30 polypeptide, a histone fold domain (TAF 12-HFD) polypeptide of human transcription factor TAF12, a cleavable self-aggregation tag INTEIN-ELK16, an E.coli maltose binding protein, an E.coli RNAse II polypeptide, an E.coli alkaline phosphatase polypeptide, an E.coli phospholipase A polypeptide, an E.coli β -lactamase polypeptide, a Salmonella typhimurium (Salmonella typhimurium) MalK protein, a C.thermocellum (Clostridium thermocellum) endoglucanase D polypeptide, a Bacillus thuringiensis aizawai subsp (Bacillus thuringiensis subsp aizawai) IPL7 insecticidal protein, a human tissue zymogen B polypeptide, a porcine interferon-gamma polypeptide, a T5 DNA polymerase polypeptide, and an E.coli thioredoxin polypeptide.

Aspect 5 the method of any one of aspects 1-4, wherein the fusion polypeptide comprises 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, 15 or more oligopeptides, 20 or more oligopeptides.

Aspect 6 the method of any one of aspects 1-5, wherein the two or more oligopeptides are not identical.

Aspect 7 the method of any one of aspects 1-6, wherein all of the oligopeptides are operably linked to the N-terminus of the insoluble carrier polypeptide or all of the oligopeptides are operably linked to the C-terminus of the insoluble carrier polypeptide.

Aspect 8 the method of any one of aspects 1-6, wherein at least one oligopeptide is operably linked to the N-terminus of the insoluble carrier polypeptide and at least one oligopeptide is operably linked to the C-terminus of the insoluble carrier polypeptide.

Aspect 9 the method of aspect 8, wherein the fusion polypeptide comprises 2 or more oligopeptides, 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides operably linked to the N-terminus of the insoluble carrier polypeptide, and/or 2 or more oligopeptides, 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides operably linked to the C-terminus of the insoluble carrier polypeptide.

Aspect 10 the method according to any one of aspects 1-9, wherein the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is with formic acid, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is with NTCB.

Aspect 11 the method according to any one of aspects 1-9, wherein the peptide bond comprises an Asp-Pro bond and the sequence specific chemical cleavage is performed with acetic acid.

Aspect 12 the method of any one of aspects 1-11, wherein the oligopeptides are operably linked by peptide bonds and released from each other when the oligopeptides are released from the insoluble carrier polypeptide.

Aspect 13 the method according to any one of aspects 1-11, wherein the oligopeptides are operably linked by different peptide bonds and released from each other by sequence-specific chemical cleavage of the different peptide bonds after the oligopeptides are released from the insoluble carrier polypeptide.

Aspect 14 the method according to aspect 13, wherein the different peptide bonds comprise (i) methionine and the sequence specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the sequence specific chemical cleavage is with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence specific chemical cleavage is with formic acid, (iv) asparagine-glycine bond and the sequence specific chemical cleavage is with hydroxylamine, or (v) cysteine and the sequence specific chemical cleavage is with NTCB.

Aspect 15 the method of aspect 13, wherein the different peptide bond comprises an Asp-Pro bond and the sequence specific chemical cleavage is performed with acetic acid.

Aspect 16 the method of any one of aspects 1-15, wherein the oligopeptide is at least 4 amino acids long, at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long, at least 15 amino acids long, at least 20 amino acids long, or at least 25 amino acids long.

Aspect 17 the method of any one of aspects 1-16, wherein the oligopeptide is less than 50 amino acids long, less than 45 amino acids long, less than 40 amino acids long, less than 35 amino acids long, less than 30 amino acids long, less than 25 amino acids long, or less than 20 amino acids long.

Aspect 18 the method of any one of aspects 1-15, wherein the oligopeptide is 4 amino acids long to 50 amino acids long, 6 amino acids long to 40 amino acids long, 6 amino acids long to 30 amino acids long or 8 amino acids long to 25 amino acids long.

Aspect 19 the method of any one of aspects 1-18, wherein the fusion peptide is expressed in bacteria or yeast.

Aspect 20 the method of aspect 19, wherein the bacterium is E.coli or Vibrio natrii (Vibrio natriegens).

Aspect 21 the method of aspect 19 or aspect 20, wherein the yield of the released oligopeptides is at least 10 mg/L of bacterial culture, at least 20 mg/L of bacterial culture, at least 30 mg/L of bacterial culture, at least 40 mg/L of bacterial culture, at least 50 mg/L of bacterial culture, at least 1 g/L or at least 5 g/L.

Aspect 22: a fusion polypeptide comprising an insoluble carrier polypeptide operably linked to two or more oligopeptides, wherein the operable linkage between the two or more oligopeptides and the insoluble carrier polypeptide comprises a peptide bond capable of sequence-specific chemical cleavage.

Aspect 23 the fusion polypeptide of aspect 22, wherein the insoluble carrier polypeptide comprises a ranpirnase polypeptide.

Aspect 24 the fusion polypeptide of aspect 22, wherein the insoluble carrier polypeptide comprises a TAF12 polypeptide.

Aspect 25 the fusion polypeptide of aspect 22, wherein the insoluble carrier polypeptide comprises a trpΔLE polypeptide, a ketosteroid isomerase (KSI) polypeptide, a β -galactosidase polypeptide, a PagP polypeptide, a truncated E.coli PurF F fragment polypeptide, a P.aeruginosa PaP3.30 polypeptide, a histone fold domain (TAF 12-HFD) polypeptide of human transcription factor TAF12, a cleavable self-aggregation tag INTEIN-ELK16, an E.coli maltose binding protein, an E.coli RNAse II polypeptide, an E.coli alkaline phosphatase polypeptide, an E.coli phospholipase A polypeptide, an E.coli β -lactamase polypeptide, a Salmonella typhimurium MalK protein, a C.thermocellum endoglucanase D polypeptide, a E.thuringiensis subspecies aizawai IPL insecticidal protein, a human tissue zymogen B polypeptide, a porcine interferon-gamma polypeptide, a T5 DNA polymerase polypeptide, and an E.coli thioredoxin polypeptide.

Aspect 26 the fusion polypeptide according to any one of aspects 22-25, wherein the fusion polypeptide comprises 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, 15 or more oligopeptides, 20 or more oligopeptides.

Aspect 27 the fusion polypeptide of any one of aspects 22-26, wherein the two or more oligopeptides are not identical.

Aspect 28 the fusion polypeptide of any one of aspects 22-27, wherein all of the oligopeptides are operably linked to the N-terminus of the insoluble carrier polypeptide or all of the oligopeptides are operably linked to the C-terminus of the insoluble carrier polypeptide.

Aspect 29 the fusion polypeptide of any one of aspects 22-27, wherein at least one oligopeptide is operably linked to the N-terminus of the insoluble carrier polypeptide and at least one oligopeptide is operably linked to the C-terminus of the insoluble carrier polypeptide.

Aspect 30 the fusion polypeptide of aspect 29, wherein the fusion polypeptide comprises 2 or more oligopeptides, 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides operably linked to the N-terminus of the insoluble carrier polypeptide, and/or 2 or more oligopeptides, 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides operably linked to the C-terminus of the insoluble carrier polypeptide.

Aspect 31 the fusion polypeptide according to any one of aspects 22-30, wherein the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is with formic acid, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is with NTCB.

Aspect 32 the fusion polypeptide according to any one of aspects 22-30, wherein the peptide bond comprises an Asp-Pro bond and the sequence specific chemical cleavage is performed with acetic acid.

Aspect 33 the fusion polypeptide according to any one of aspects 22-32, wherein the oligopeptides are operably linked by the peptide bond and are capable of being released from each other by the sequence-specific chemical cleavage.

Aspect 34 the fusion polypeptide according to any one of aspects 22-32, wherein the oligopeptides are operably linked by different peptide bonds and are capable of being released from each other by different sequence-specific chemical cleavage.

Aspect 35 the fusion polypeptide according to aspect 34, wherein the different peptide bond comprises (i) methionine and the different sequence-specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the different sequence-specific chemical cleavage is with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the different sequence-specific chemical cleavage is with formic acid, (iv) asparagine-glycine bond and the different sequence-specific chemical cleavage is with hydroxylamine, or (ntcv) cysteine and the different sequence-specific chemical cleavage is with NTCB.

Aspect 36 the fusion polypeptide according to aspect 34, wherein the different peptide bond comprises an Asp-Pro bond and the different sequence-specific chemical cleavage is performed with acetic acid.

Aspect 37 the fusion polypeptide according to any one of aspects 22-36, wherein the oligopeptide is at least 4 amino acids long, at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long, at least 15 amino acids long, at least 20 amino acids long or at least 25 amino acids long.

Aspect 38 the fusion polypeptide according to any one of aspects 22-37, wherein the oligopeptide is less than 50 amino acids long, less than 45 amino acids long, less than 40 amino acids long, less than 35 amino acids long, less than 30 amino acids long, less than 25 amino acids long or less than 20 amino acids long.

Aspect 39 the fusion polypeptide according to any one of aspects 22-36, wherein the oligopeptide is 4 amino acids long to 50 amino acids long, 6 amino acids long to 40 amino acids long, 6 amino acids long to 30 amino acids long or 8 amino acids long to 25 amino acids long.

Aspect 40A method of releasing an oligopeptide fused to an insoluble carrier polypeptide that forms inclusion bodies in a cell, comprising

A) Expressing a fusion polypeptide comprising said oligopeptide operably linked to said insoluble carrier polypeptide, wherein said operable linkage is a peptide bond capable of sequence specific chemical cleavage by acetic acid,

B) Purifying the inclusion bodies, and

C) Incubating the inclusion bodies with an acid at a temperature of greater than 50 ℃ for at least 1 hour, wherein the oligopeptide is released from the fusion polypeptide by sequence specific cleavage of the peptide bond.

Aspect 41 the method of aspect 40, wherein the insoluble carrier polypeptide comprises a ranpirnase polypeptide.

Aspect 42 the method of aspect 40, wherein the insoluble carrier polypeptide comprises a TAF12 polypeptide.

Aspect 43 the method of aspect 40, wherein the insoluble carrier polypeptide comprises a trpΔLE polypeptide, a ketosteroid isomerase (KSI) polypeptide, a β -galactosidase polypeptide, a PagP polypeptide, a truncated E.coli PurF F fragment polypeptide, a P.aeruginosa PaP3.30 polypeptide, a histone fold domain (TAF 12-HFD) polypeptide of human transcription factor TAF12, a cleavable self-aggregation tag INTEIN-ELK16, an E.coli maltose binding protein, an E.coli RNAse II polypeptide, an E.coli alkaline phosphatase polypeptide, an E.coli phospholipase A polypeptide, an E.coli β -lactamase polypeptide, a Salmonella typhimurium MalK protein, a C.thermocellum endoglucanase D polypeptide, a E.thuringiensis subspecies aizawai IPL7 insecticidal protein, a human tissue zymogen B polypeptide, a porcine interferon-gamma polypeptide, a T5 DNA polymerase polypeptide, and an E.coli thioredoxin polypeptide.

Aspect 44 the method of any one of aspects 40-43, wherein the temperature of step C) is greater than 60 ℃, greater than 70 ℃, greater than 80 ℃, or greater than 90 ℃.

Aspect 45 the method of any one of aspects 40-44, wherein the temperature of step C) is less than 100 ℃, less than 95 ℃, less than 90 ℃, or less than 85 ℃.

Aspect 46 the method of any one of aspects 40-45, wherein the pH in step c) is below 3.0.

Aspect 47 the method of any one of aspects 40-45, wherein the pH in step c) is from 2.6 to 2.8.

Aspect 48 the method of any one of aspects 40-47, wherein the acid is a strong acid, optionally selected from hydrochloric acid and sulfuric acid.

Aspect 49 the method of any one of aspects 40-47, wherein the acid is a weak acid.

Aspect 50 the method of aspect 49, wherein the weak acid is acetic acid.

Aspect 51 the method of aspect 50, wherein the concentration of acetic acid is at least 2 wt.%, at least 3 wt.%, at least 4 wt.%, or at least 5 wt.%.

Aspect 52 the method of aspect 50 or aspect 51, wherein the concentration of acetic acid is less than 50 wt.%, less than 45 wt.%, less than 40 wt.%, less than 35 wt.%, or less than 30 wt.%.

Aspect 53 the method of any one of aspects 40-52, wherein the insoluble carrier polypeptide is solubilized by the incubation of step c).

Aspect 54 the method of any one of aspects 40-52, wherein the insoluble carrier polypeptide is not solubilized by the incubation of step c).

Aspect 55 the method of any one of aspects 40-54, wherein the fusion polypeptide comprises 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, 15 or more oligopeptides, 20 or more oligopeptides.

Aspect 56 the method of any one of aspects 40-55, wherein the two or more oligopeptides are not identical.

Aspect 57 the method of any one of aspects 40-56, wherein all of the oligopeptides are operably linked to the N-terminus of the insoluble carrier polypeptide or all of the oligopeptides are operably linked to the C-terminus of the insoluble carrier polypeptide.

Aspect 58 the method of any one of aspects 40-56, wherein at least one oligopeptide is operably linked to the N-terminus of the insoluble carrier polypeptide and at least one oligopeptide is operably linked to the C-terminus of the insoluble carrier polypeptide.

Aspect 59 the method of aspect 58, wherein the fusion polypeptide comprises 2 or more oligopeptides, 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides operably linked to the N-terminus of the insoluble carrier polypeptide, and/or 2 or more oligopeptides, 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides operably linked to the C-terminus of the insoluble carrier polypeptide.

Aspect 60 the method of any one of aspects 40-59, wherein the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is with formic acid, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is with NTCB.

Aspect 61 the method of any one of aspects 40-60, wherein the peptide bond comprises an Asp-Pro bond and the sequence specific chemical cleavage is acid cleavage.

Aspect 62 the method of any one of aspects 40-61, wherein the oligopeptides are operably linked by peptide bonds and released from each other when the oligopeptides are released from the insoluble carrier polypeptide.

Aspect 63 the method of any one of aspects 40-62, wherein the oligopeptides are operably linked by different peptide bonds and released from each other by sequence-specific chemical cleavage of the different peptide bonds after the oligopeptides are released from the insoluble carrier polypeptide.

Aspect 64 the method of aspect 63, wherein the different peptide bonds comprise (i) methionine and the sequence-specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is with formic acid, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is with NTCB.

Aspect 65 the method of aspect 64, wherein said different peptide bond comprises an Asp-Pro bond and said sequence specific chemical cleavage is acid cleavage.

Aspect 66 the method of any one of aspects 40-65, wherein the oligopeptide is at least 4 amino acids long, at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long, at least 15 amino acids long, at least 20 amino acids long, or at least 25 amino acids long.

Aspect 67 the method of any one of aspects 40-66, wherein the oligopeptide is less than 50 amino acids long, less than 45 amino acids long, less than 40 amino acids long, less than 35 amino acids long, less than 30 amino acids long, less than 25 amino acids long, or less than 20 amino acids long.

Aspect 68 the method of any one of aspects 40-65, wherein the oligopeptide is 4 amino acids long to 50 amino acids long, 6 amino acids long to 40 amino acids long, 6 amino acids long to 30 amino acids long, or 8 amino acids long to 25 amino acids long.

Aspect 69 the method of any one of aspects 40-68, wherein the fusion peptide is expressed in bacteria or yeast.

Aspect 70 the method of aspect 69, wherein the bacterium is E.coli or Vibrio natrii (Vibrio natriegens).

Aspect 71A method of purifying inclusion bodies comprising a fusion protein, the method comprising

A) Expressing a fusion protein comprising an oligopeptide operatively linked to an insoluble carrier polypeptide that forms inclusion bodies in a cell, wherein said operative linkage is a chemically cleavable amino acid sequence,

B) Lysing the cells to form a cell lysate,

C) Centrifuging the cell lysate to form a pellet,

D) Washing the precipitate at least once, at least twice or at least three times in a surfactant buffer comprising a nonionic surfactant,

E) Washing the precipitate at least once, at least twice or at least three times in a salt buffer comprising at least 0.5M NaCl, and

F) Washing the pellet at least once, at least twice or at least three times in water, thereby producing purified inclusion bodies.

Aspect 72 the method of aspect 71, wherein the insoluble carrier polypeptide comprises a TAF12 polypeptide.

Aspect 73 the method of aspect 71, wherein the insoluble carrier polypeptide comprises a ranpirnase polypeptide.

Aspect 74 the method of aspect 71, wherein the insoluble carrier polypeptide comprises a trpΔLE polypeptide, a ketosteroid isomerase (KSI) polypeptide, a β -galactosidase polypeptide, a PagP polypeptide, a truncated E.coli PurF F fragment polypeptide, a P.aeruginosa PaP3.30 polypeptide, a histone fold domain (TAF 12-HFD) polypeptide of human transcription factor TAF12, a cleavable self-aggregation tag INTEIN-ELK16, an E.coli maltose binding protein, an E.coli RNAse II polypeptide, an E.coli alkaline phosphatase polypeptide, an E.coli phospholipase A polypeptide, an E.coli β -lactamase polypeptide, a Salmonella typhimurium MalK protein, a C.thermocellum endoglucanase D polypeptide, a E.thuringiensis subspecies aizawai IPL7 insecticidal protein, a human tissue zymogen B polypeptide, a porcine interferon-gamma polypeptide, a T5 DNA polymerase polypeptide, and an E.coli thioredoxin polypeptide.

Aspect 75 the method of any one of aspects 71-74, wherein the salt buffer is at least 0.6M NaCl, at least 0.7M NaCl, or at least 0.75M NaCl.

Aspect 76 the method of any one of aspects 71-75, wherein the fusion polypeptide comprises 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, 15 or more oligopeptides, 20 or more oligopeptides.

Aspect 77 the method of any one of aspects 71-76, wherein the two or more oligopeptides are not identical.

Aspect 78 the method of any one of aspects 71-77, wherein all of the oligopeptides are operably linked to the N-terminus of the insoluble carrier polypeptide or all of the oligopeptides are operably linked to the C-terminus of the insoluble carrier polypeptide.

Aspect 79 the method of any one of aspects 71-77, wherein at least one oligopeptide is operably linked to the N-terminus of the insoluble carrier polypeptide and at least one oligopeptide is operably linked to the C-terminus of the insoluble carrier polypeptide.

Aspect 80 the method of aspect 79, wherein the fusion polypeptide comprises 2 or more oligopeptides, 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides operably linked to the N-terminus of the insoluble carrier polypeptide, and/or 2 or more oligopeptides, 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides operably linked to the C-terminus of the insoluble carrier polypeptide.

Aspect 81 the method of any one of aspects 71-80, wherein the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is with formic acid, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is with NTCB.

Aspect 82 the method of any one of aspects 71-80, wherein the peptide bond comprises an Asp-Pro bond and the sequence specific chemical cleavage is acid cleavage.

Aspect 83 the method of any one of aspects 71-82, wherein the oligopeptides are operably linked by peptide bonds and released from each other when the oligopeptides are released from the insoluble carrier polypeptide.

Aspect 84 the method of any one of aspects 71-82, wherein the oligopeptides are operably linked by different peptide bonds and released from each other by sequence-specific chemical cleavage of the different peptide bonds after the oligopeptides are released from the insoluble carrier polypeptide.

Aspect 85 the method of aspect 84, wherein the different peptide bonds comprise (i) methionine and the sequence-specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is with formic acid, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is with NTCB.

Aspect 86 the method of aspect 84, wherein said different peptide bond comprises an Asp-Pro bond and said sequence specific chemical cleavage is performed with acetic acid.

Aspect 87 the method of any one of aspects 71-86, wherein the oligopeptide is at least 4 amino acids long, at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long, at least 15 amino acids long, at least 20 amino acids long, or at least 25 amino acids long.

Aspect 88 the method of any one of aspects 71-87, wherein the oligopeptide is less than 50 amino acids long, less than 45 amino acids long, less than 40 amino acids long, less than 35 amino acids long, less than 30 amino acids long, less than 25 amino acids long, or less than 20 amino acids long.

Aspect 89 the method of any one of aspects 71-86, wherein the oligopeptide is 4 amino acids long to 50 amino acids long, 6 amino acids long to 40 amino acids long, 6 amino acids long to 30 amino acids long, or 8 amino acids long to 25 amino acids long.

Aspect 90 the method of any one of aspects 71-89, wherein the fusion peptide is expressed in bacteria or yeast.

Aspect 91 the method of aspect 90, wherein the bacterium is E.coli or Vibrio natrii (Vibrio natriegens).

Aspect 92: a method of producing a fusion polypeptide comprising expressing a fusion polypeptide comprising an oligopeptide operably linked to the C-terminus of a ranpirnase polypeptide, wherein the ranpirnase polypeptide comprises one or more amino acid substitutions in 11N-terminal amino acids as compared to SEQ ID NO:1, wherein the ranpirnase-oligopeptide fusion protein comprising the ranpirnase polypeptide is expressed at a higher level of expression than the ranpirnase fusion protein comprising SEQ ID NO:1 when expressed under the same conditions.

Aspect 93 the method of aspect 92, wherein the fusion polypeptide comprises 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, 15 or more oligopeptides, 20 or more oligopeptides.

Aspect 94 the method of aspect 92 or aspect 95, wherein the two or more oligopeptides are not identical.

Aspect 95 the fusion polypeptide of any one of aspects 92-94, wherein all of the oligopeptides are operably linked to the N-terminus of the ranpirnase polypeptide or all of the oligopeptides are operably linked to the C-terminus of the ranpirnase polypeptide.

Aspect 96 the method of any one of aspects 92-94, wherein at least one oligopeptide is operably linked to the N-terminus of the ranpirnase polypeptide and at least one oligopeptide is operably linked to the C-terminus of the ranpirnase polypeptide.

Aspect 97 the method of aspect 96, wherein the fusion polypeptide comprises 2 or more oligopeptides, 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides operably linked to the N-terminus of the ranpirnase polypeptide, and/or 2 or more oligopeptides, 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides operably linked to the C-terminus of the ranpirnase polypeptide.

Aspect 98 the method of any one of aspects 92-97, wherein the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is with formic acid, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is with NTCB.

Aspect 99 the method of any one of aspects 92-97, wherein the peptide bond comprises an Asp-Pro bond and the sequence specific chemical cleavage is performed with acetic acid.

Aspect 100 the method of any one of aspects 92-99, wherein the oligopeptides are operably linked by peptide bonds and released from each other when the oligopeptides are released from the ranpirnase.

Aspect 101 the method of any one of aspects 92-99, wherein the oligopeptides are operably linked by different peptide bonds and released from each other by sequence-specific chemical cleavage of the different peptide bonds after the oligopeptides are released from the ranpirnase.

Aspect 102 the method of aspect 101, wherein the different peptide bonds comprise (i) methionine and the sequence-specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is with formic acid, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is with NTCB.

Aspect 103 the method of aspect 101, wherein said different peptide bond comprises an Asp-Pro bond and said sequence specific chemical cleavage is acid cleavage.

Aspect 104 the method of any one of aspects 92-103, wherein the oligopeptide is at least 4 amino acids long, at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long, at least 15 amino acids long, at least 20 amino acids long, or at least 25 amino acids long.

Aspect 105 the method of any one of aspects 92-104, wherein the oligopeptide is less than 50 amino acids long, less than 45 amino acids long, less than 40 amino acids long, less than 35 amino acids long, less than 30 amino acids long, less than 25 amino acids long, or less than 20 amino acids long.

Aspect 106 the method of any one of aspects 92-103, wherein the oligopeptide is 4 amino acids long to 50 amino acids long, 6 amino acids long to 40 amino acids long, 6 amino acids long to 30 amino acids long, or 8 amino acids long to 25 amino acids long.

Aspect 107 the method of any one of aspects 92-106, wherein the fusion peptide is expressed in bacteria or yeast.

Aspect 108 the method of aspect 107, wherein the bacterium is E.coli or Vibrio natrii (Vibrio natriegens).

Aspect 109: a ranpirnase polypeptide comprising one or more amino acid substitutions in the 11N-terminal amino acids of the ranpirnase polypeptide as compared to SEQ ID No. 1, wherein the ranpirnase polypeptide is expressed at a higher level of expression than the ranpirnase protein of SEQ ID No. 1 when expressed under the same conditions.

Aspect 110 the ranpirnase polypeptide of aspect 109 wherein the ranpirnase polypeptide comprises the amino acid sequence of one of SEQ ID NOs 2-10 and 15-22, optionally with 1,2, 3,4, 5 or all 6C-terminal histidine residues deleted, as shown in figure 20A.

Aspect 111 a fusion polypeptide comprising a ranpirnase polypeptide operably linked to one or more oligopeptides, wherein the operable linkage between the one or more oligopeptides and the ranpirnase polypeptide comprises a peptide bond capable of sequence-specific chemical cleavage, wherein the ranpirnase polypeptide comprises one or more amino acid substitutions in the 11N-terminal amino acids of the ranpirnase polypeptide compared to SEQ ID No. 1, wherein the ranpirnase polypeptide is expressed at a higher level of expression than the ranpirnase protein of SEQ ID No. 1 when expressed under the same conditions.

Aspect 112 the fusion polypeptide of aspect 111, wherein the ranpirnase polypeptide comprises the amino acid sequence of one of SEQ ID NOs 2-10 and 15-22, optionally with 1, 2, 3,4, 5, or all 6C-terminal histidine residues deleted, as shown in figure 20A.

Aspect 113 the fusion polypeptide according to aspect 111 or aspect 112, wherein the fusion polypeptide comprises 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, 15 or more oligopeptides, 20 or more oligopeptides.

Aspect 114 the fusion polypeptide of any one of aspects 111-113, wherein the two or more oligopeptides are not identical.

Aspect 115 the fusion polypeptide of any one of aspects 111-114, wherein all of the oligopeptides are operably linked to the N-terminus of the ranpirnase polypeptide or all of the oligopeptides are operably linked to the C-terminus of the ranpirnase polypeptide.

Aspect 116 the fusion polypeptide of any one of aspects 111-114, wherein at least one oligopeptide is operably linked to the N-terminus of the ranpirnase polypeptide and at least one oligopeptide is operably linked to the C-terminus of the ranpirnase polypeptide.

Aspect 117 is the fusion polypeptide of aspect 116, wherein the fusion polypeptide comprises 2 or more oligopeptides, 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, and/or 2 or more oligopeptides, 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 10 or more oligopeptides operably linked to the C-terminus of the ranpirnase polypeptide.

Aspect 118 the fusion polypeptide according to any one of aspects 111-117, wherein the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is with formic acid, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is with NTCB.

Aspect 119 the fusion polypeptide of any one of aspects 111-117, wherein the peptide bond comprises an Asp-Pro bond and the sequence specific chemical cleavage is acid cleavage.

Aspect 120 the fusion polypeptide according to any one of aspects 111-119, wherein the oligopeptides are operably linked by the peptide bond and are capable of being released from each other by the sequence-specific chemical cleavage.

Aspect 121 the fusion polypeptide of any one of aspects 105-119, wherein the oligopeptides are operably linked by different peptide bonds and are capable of being released from each other by different sequence-specific chemical cleavage.

Aspect 122 the fusion polypeptide of aspect 121, wherein the different peptide bond comprises (i) methionine and the different sequence-specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the different sequence-specific chemical cleavage is with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the different sequence-specific chemical cleavage is with formic acid, (iv) asparagine-glycine bond and the different sequence-specific chemical cleavage is with hydroxylamine, or (ntcv) cysteine and the different sequence-specific chemical cleavage is with NTCB.

Aspect 123 the fusion polypeptide of aspect 121, wherein the different peptide bond comprises an Asp-Pro bond and the different sequence-specific chemical cleavage is acid cleavage.

Aspect 124 the fusion polypeptide of any one of aspects 111-123, wherein the oligopeptide is at least 4 amino acids long, at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long, at least 15 amino acids long, at least 20 amino acids long, or at least 25 amino acids long.

Aspect 125 the fusion polypeptide of any one of aspects 111-124, wherein the oligopeptide is less than 50 amino acids long, less than 45 amino acids long, less than 40 amino acids long, less than 35 amino acids long, less than 30 amino acids long, less than 25 amino acids long, or less than 20 amino acids long.

Aspect 126 the fusion polypeptide of any one of aspects 111-125, wherein the oligopeptide is 4 amino acids long to 50 amino acids long, 6 amino acids long to 40 amino acids long, 6 amino acids long to 30 amino acids long, or 8 amino acids long to 25 amino acids long.

Aspect 127 an oligopeptide comprising an active amino acid sequence and N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid, wherein the active amino acid sequence is miPEP and the oligopeptide-modulating miRNA or the active amino acid sequence is a peptide microbial inhibitor and the oligopeptide inhibits a microorganism.

Aspect 128 is a nucleic acid encoding the fusion polypeptide according to any one of aspects 22-39, the ranpirnase according to aspect 109 or aspect 110, or the fusion polypeptide according to any one of aspects 111-126.

Aspect 129 a cell comprising a nucleic acid according to aspect 128.

Aspect 130 the cell of aspect 129, wherein the cell is a bacterial cell or a yeast cell.

Aspect 131 the cell of aspect 129, wherein the bacterium is E.coli or Vibrio natrii.

Aspect 132 the cell of aspect 131, wherein the cell is a BL21 bacterial cell.

Aspect 133 the cell of aspect 131, wherein the cell does not express Lon and ompT proteases.

Aspect 134 the nucleic acid of aspect 128, wherein the nucleic acid is an isolated nucleic acid.

Aspect 135 the method of any one of aspects 1-108, wherein the fusion polypeptide or fusion peptide is expressed in E.coli.

Aspect 136 the method of any one of aspects 1-108 and 135, wherein the fusion polypeptide or fusion protein is expressed in a cell grown in a fermentation bioreactor.

Aspect 137 the method of any one of aspects 1-108, wherein the cutting is performed at a pH of about 2-3.5 and a temperature of about 70-90 ℃ for about 1-24 hours.

Aspect 138 is a method of producing a fusion polypeptide comprising expressing a fusion polypeptide comprising an oligopeptide operably linked to the C-terminus or C-terminus of a modified TAF polypeptide, wherein the modified TAF polypeptide comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 23.

Aspect 139 the method of aspect 138, wherein the fusion polypeptide comprises 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, 15 or more oligopeptides, 20 or more oligopeptides.

Aspect 140 the method of aspect 138 or aspect 139, wherein the two or more oligopeptides are not identical.

Aspect 141 the method of any one of aspects 138-140, wherein all of the oligopeptides are operably linked to the N-terminus of the modified TAF polypeptide or all of the oligopeptides are operably linked to the C-terminus of the modified TAF polypeptide.

Aspect 142 the method of any one of aspects 138-140, wherein at least one oligopeptide is operably linked to the N-terminus of the modified TAF polypeptide and at least one oligopeptide is operably linked to the C-terminus of the modified TAF polypeptide.

Aspect 143 the method of aspect 142, wherein the fusion polypeptide comprises 2 or more oligopeptides, 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides operably linked to the N-terminus of the modified TAF polypeptide, and/or 2 or more oligopeptides, 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides operably linked to the C-terminus of the modified TAF polypeptide.

Aspect 144 the method of any one of aspects 138-143, wherein the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is with formic acid, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is with NTCB.

Aspect 145 the method of any one of aspects 138-143, wherein the peptide bond comprises an Asp-Pro bond and the sequence specific chemical cleavage is performed with acetic acid or sulfuric acid.

Aspect 146 the method of any one of aspects 138-145, wherein the oligopeptides are operably linked by peptide bonds and released from each other when the oligopeptides are released from the modified TAF polypeptide.

Aspect 147 the method of any one of aspects 138-145, wherein the oligopeptides are operably linked by different peptide bonds and released from each other by sequence-specific chemical cleavage of the different peptide bonds after the oligopeptides are released from the modified TAF polypeptide.

Aspect 148 the method of aspect 147, wherein the different peptide bonds comprise (i) methionine and the sequence-specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is with formic acid, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is with NTCB.

Aspect 149 the method of aspect 147, wherein the different peptide bond comprises an Asp-Pro bond and the sequence specific chemical cleavage is acid cleavage.

Aspect 150 the method of any one of aspects 138-149, wherein the oligopeptide is at least 4 amino acids long, at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long, at least 15 amino acids long, at least 20 amino acids long, or at least 25 amino acids long.

Aspect 151 the method of any one of aspects 138-150, wherein the oligopeptide is less than 50 amino acids long, less than 45 amino acids long, less than 40 amino acids long, less than 35 amino acids long, less than 30 amino acids long, less than 25 amino acids long, or less than 20 amino acids long.

Aspect 152 the method of any one of aspects 138-149, wherein the oligopeptide is 4 amino acids long to 50 amino acids long, 6 amino acids long to 40 amino acids long, 6 amino acids long to 30 amino acids long, or 8 amino acids long to 25 amino acids long.

Aspect 153 the method of any one of aspects 138-152, wherein the fusion peptide is expressed in bacteria or yeast.

Aspect 154 the method of aspect 153, wherein the bacterium is E.coli or Vibrio natrii (Vibrio natriegens).

Aspect 155 is a modified TAF polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO. 23.

Aspect 156 the modified TAF polypeptide of aspect 155, wherein said modified TAF polypeptide comprises the amino acid sequence of SEQ ID No. 23.

Aspect 157: a fusion polypeptide comprising a modified TAF polypeptide operably linked to one or more oligopeptides, wherein the operable linkage between the one or more oligopeptides and the modified TAF polypeptide comprises a peptide bond capable of sequence-specific chemical cleavage, wherein the modified TAF polypeptide comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 23.

Aspect 158 the fusion polypeptide of aspect 157, wherein the modified TAF polypeptide comprises the amino acid sequence of SEQ ID NO. 23.

Aspect 159 the fusion polypeptide of aspect 157 or aspect 158, wherein the fusion polypeptide comprises 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, 15 or more oligopeptides, 20 or more oligopeptides.

Aspect 160 the fusion polypeptide of any one of aspects 157-159, wherein the two or more oligopeptides are not identical.

Aspect 161 the fusion polypeptide of any of aspects 157-160, wherein all of the oligopeptides are operably linked to the N-terminus of the modified TAF polypeptide or all of the oligopeptides are operably linked to the C-terminus of the modified TAF polypeptide.

Aspect 162 the fusion polypeptide of any one of aspects 157-160, wherein at least one oligopeptide is operably linked to the N-terminus of the modified TAF polypeptide and at least one oligopeptide is operably linked to the C-terminus of the modified TAF polypeptide.

Aspect 163 the fusion polypeptide of aspect 162, wherein the fusion polypeptide comprises 2 or more oligopeptides, 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, and/or 2 or more oligopeptides, 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 10 or more oligopeptides operably linked to the C-terminus of the modified TAF polypeptide.

Aspect 164 the fusion polypeptide according to any one of aspects 157-163, wherein the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is with formic acid, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is with NTCB.

Aspect 165 the fusion polypeptide according to any one of aspects 157-163, wherein the peptide bond comprises an Asp-Pro bond and the sequence specific chemical cleavage is acid cleavage.

Aspect 166 the fusion polypeptide according to any one of aspects 157-165, wherein the oligopeptides are operably linked by the peptide bond and are capable of being released from each other by the sequence-specific chemical cleavage.

Aspect 167 the fusion polypeptide of any of aspects 157-165, wherein the oligopeptides are operably linked by different peptide bonds and are capable of release from each other by different sequence-specific chemical cleavage.

Aspect 168: the fusion polypeptide according to aspect 167, wherein the different peptide bond comprises (i) methionine and the different sequence-specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the different sequence-specific chemical cleavage is with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the different sequence-specific chemical cleavage is with formic acid, (iv) asparagine-glycine bond and the different sequence-specific chemical cleavage is with hydroxylamine, or (ntcv) cysteine and the different sequence-specific chemical cleavage is with NTCB.

Aspect 169 the fusion polypeptide according to aspect 167, wherein said different peptide bond comprises an Asp-Pro bond and said different sequence-specific chemical cleavage is acid cleavage.

Aspect 170 the fusion polypeptide of any one of aspects 157-169, wherein the oligopeptide is at least 4 amino acids long, at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long, at least 15 amino acids long, at least 20 amino acids long, or at least 25 amino acids long.

Aspect 171 the fusion polypeptide of any one of aspects 157-170, wherein the oligopeptide is less than 50 amino acids long, less than 45 amino acids long, less than 40 amino acids long, less than 35 amino acids long, less than 30 amino acids long, less than 25 amino acids long, or less than 20 amino acids long.

Aspect 172 the fusion polypeptide of any one of aspects 157-171, wherein the oligopeptide is 4 amino acids long to 50 amino acids long, 6 amino acids long to 40 amino acids long, 6 amino acids long to 30 amino acids long, or 8 amino acids long to 25 amino acids long.

Aspect 173 a nucleic acid encoding the fusion polypeptide of any one of aspects 22-39, the modified RAF polypeptide of aspect 155 or aspect 156, or the fusion polypeptide of any one of aspects 157-172.

Aspect 174 is a cell comprising the nucleic acid of aspect 173.

Aspect 175 the cell of aspect 174, wherein the cell is a bacterial cell or a yeast cell.

Aspect 176 the cell of aspect 174, wherein the bacterium is E.coli or Vibrio natrii.

Aspect 177 the cell of aspect 176, wherein the cell is a BL21 bacterial cell.

Aspect 178 the cell of aspect 176, wherein the cell does not express Lon and ompT proteases.

Aspect 179 the nucleic acid of aspect 173, wherein the nucleic acid is an isolated nucleic acid.

Drawings

FIG. 1A is a schematic representation of a ranpirnase-based fusion construct for use in peptide bio-production strategies as an exemplary means of producing peptides with insoluble carrier polypeptides. FIG. 1B shows RP-HPLC chromatograms of four pH's tested in the first experiment of example 1, with overlapping peak positions at the highlighted peptide and protein fractions.

Figures 2A-2B compare the amounts of peptide and fusion protein released at four pH tested from pH 2 to pH 5. Figure 2A shows the amount of peptide released by cleavage at the four pH tested. Figure 2B shows the amount of fusion protein solubilized at the four pH's tested.

Figures 3A-3B compare the amounts of peptide and fusion protein released at seven pH tested from pH 1.5 to pH 3. Figure 3A shows the amount of peptide released by cleavage at seven pH tested. Figure 3B shows the amount of fusion protein solubilized at the seven pH tested.

Figures 4A-4B show an increase in the amount of acidic form of peptide at a decrease in pH. FIG. 4A shows an overlapping RP-HPLC chromatogram for each of the 5 pH's tested, with the alkaline form on the left and the acidic form on the right. Fig. 4B shows the relative amounts of alkaline form (black bars) and acidic form (grey bars) at the 8 pH tested.

FIGS. 5A-5B show SDS PAGE of the tested fusion protein constructs and the respective post-expression cell lysates. Fig. 5A shows the five constructs tested. FIG. 5B shows SDS PAGE, from left to right, of molecular weight markers, ranpirnase fusions, KSI fusions, purF fusions, TAF12 fusions, and OmpX fusions. After cleavage and purification, the amounts of peptide (relative to ranpirnase as 100%) were KSI fusion (120%), purF fusion (75%), TAF12 fusion (265%) and OmpX fusion (37%).

FIGS. 6A-6C are schematic illustrations of different ranpirnase-based fusion strategies for peptide bio-production as an exemplary means of producing peptides with insoluble carrier polypeptides. FIG. 6A is a schematic of a homologous concatemer ranpirnase fusion construct in which several identical peptide sequences have additional aspartate-proline (DP) cleavage sites added and the peptides have up to 10 copies (N). FIG. 6B is a schematic of a heterologous concatemer construct in which copies of peptides of different sequences (M) were linked to Asp-Pro (DP) cleavage sites fused to ranpirnase proteins. FIG. 6C shows ranpirnase fusions with only one peptide sequence at the C-terminus.

FIG. 7 shows SDS-PAGE analysis of insoluble fractions of different ranpirnase constructs with 1 (X1) or 3 (X3) peptide copies. Peptides A, B and C are hydrophilic peptides 10 amino acids long, but differ in their amino acid sequence. MW: molecular weight markers.

FIGS. 8A-8B show cleavage results from concatemer ONC fusion proteins. FIG. 8A shows an RP-HPLC chromatogram after cleavage for 4 hours, in which the mono-, di-and tripeptide peaks are labeled. FIG. 8B shows an RP-HPLC chromatogram after 16 hours of cleavage, with mono-and dipeptide peaks labeled.

Figures 9A-9C show the results of N-terminal variants of ranpirnase and comparative peptide yield studies. FIG. 9A is a schematic representation of the ranpirnase construct (construct A (SEQ ID NO:2403)、B (SEQ ID NO:2404)、C (SEQ ID NO:2405)、D (SEQ ID NO:2406)、E (SEQ ID NO:2407)、F (SEQ ID NO:2408)、G (SEQ ID NO:2409)、H (SEQ ID NO:2410) and WT N-ter sequence (SEQ ID NO: 2411)). Figure 9B shows the sequences exchanged to generate all N-terminal sequences of the ranpirnase variant constructs. FIG. 9C shows a comparison of peptide yields obtained after chemical cleavage for ranpirnase constructs A-H and unmutated ranpirnase.

FIG. 10 shows a comparison of the quality of MP18357 peptides produced with three different ranpirnase variant constructs with modified N-terminus. If SEQ ID NO. 1 is shown in the panel insert (upper right corner of each panel), a comparison of the HPLC profile with the original ranpirnase construct is shown.

FIGS. 11A-11B show the results of chemical cleavage optimization using the concatemer ranpirnase strategy. FIG. 11A is a schematic of the ranpirnase concatemer strategy used. Peptides of different sizes are produced when incomplete chemical cleavage of the concatemers occurs. FIG. 11B shows RP-HPLC spectra of incomplete chemical digestion of ranpirnase concatamers at 40 ℃.

FIGS. 12A-12C show the time course of the percentage of peptide distribution detected by RP-HPLC after incubation with acetic acid. Chemical cleavage was performed at 40 ℃ (fig. 12A), 60 ℃ (fig. 12B) or 80 ℃ (fig. 12C). The Y-axis represents the percentage of each species (mono-, di-or tripeptides).

Figure 13 shows a comparison of peptide release over time after chemical cleavage of the concatemer ranpirnase constructs at different temperatures.

FIGS. 14A-14D show RP-HPLC analysis of concatemer constructs with 3 copies of MP18357 peptide. FIG. 14A shows RP-HPLC spectra (overlaid) obtained after solubilization at 60℃using protein concentrations of 20 g/L, and 20%, 30%, 40% and 50% acetic acid. FIG. 14B shows RP-HPLC spectra (overlaid) obtained after solubilization at 80℃using protein concentrations of 50 g/L, and 20%, 30%, 40% and 50% acetic acid. FIG. 14C shows RP-HPLC spectra (overlaid) obtained after solubilization at 60℃using protein concentrations of 20 g/L, and 20%, 30%, 40% and 50% formic acid. FIG. 14D shows a comparison of RP-HPLC spectra (overlaid) obtained after solubilization at 80℃using 50 g/L protein concentration, and 20% formic acid or 20% acetic acid.

FIGS. 15A-15B are schematic diagrams of peptide production schemes. Fig. 15A is a schematic of a chaotropic protocol for peptide production, while fig. 15B is a schematic workflow protocol requiring the addition of chaotropic agents for ranpirnase solubilization.

Figure 16 shows a comparison of peptide yields therebetween when peptide production was performed using a chaotropic agent-free protocol or a protocol requiring the addition of chaotropic agents for ranpirnase solubilization. Yield is expressed in mg/L flask culture.

Figure 17 shows the percent (%) disease control of peptides sprayed on botrytis cinerea infected tomato plants. Peptides were sprayed at a concentration of 0.1 g/l or 0.3 g/l 24 hours after infection.

Figure 18A shows the design of ranpirnase fusion constructs with 1 to 6 copies of oligopeptide MP 18913. FIG. 18B provides a table with the total number of peptides, the total number of peptides at the N-terminus, and the number of peptides at the C-terminus. FIG. 18C shows SDS-PAGE analysis of fusion protein expression using 10. Mu.L/well and shows, from left to right, ONC357_1x3, ONC357_2x3, ONC357_3x3, ONC357x3, ONC357x4, NEB P7717 molecular weight markers, ONC357x3, ONC357x4, ONC357x5, ONC357x6 and NEB P7717 molecular weight markers.

Figure 19 shows HPLC analysis of peptide release after acidic cleavage.

FIG. 20A shows amino acid sequence comparisons between SEQ ID NO 2412 and ranpirnase variants (SEQ ID NO: 2413) comprising three lysine to alanine substitutions and six C-terminal histidine deletions. FIG. 20B shows an alignment of several ranpirnase variants (SEQ ID NOS: 2414-2421).

FIGS. 21A-21B show exemplary schematic diagrams of cells and batch cultures generated by incorporating inclusion bodies into E.coli cultures. FIG. 21A shows an exemplary schematic of an E.coli cell containing a DNA concatemer comprising a fusion carrier protein (also marked with an asterisk), two amino acids that are repeated as linkers ("XY"), and a peptide of interest ("PEP") located between the linkers. FIG. 21B shows an exemplary bioreactor during the amplification of the expressed peptide titer batch (left) and E.coli cells (right) that produced inclusion bodies due to the activity of the incorporated DNA concatemers (grey dashed line).

Detailed Description

Provided herein are methods of producing oligopeptides, methods of releasing oligopeptides fused to insoluble carrier polypeptides that form inclusion bodies in cells, methods of purifying inclusion bodies comprising fusion proteins, and methods of producing fusion polypeptides. Also provided herein are fusion polypeptides comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) and a modified insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) operably linked to two or more oligopeptides comprising an active amino acid sequence.

The methods and polypeptides of the present disclosure are based, at least in part, on applicants' discovery of biological production processes utilizing concatemer protein constructs comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) and two or more oligopeptides, which improve peptide yield and peptide quality relative to other biological production methods. Applicants' biological production process surprisingly improves the production of short bioactive peptides, which are known in the art to be challenging to produce. Compared to previous methods, the methods disclosed herein do not require the use of chaotropic agents or column-based purification steps, thus reducing the economic and technical burden of peptide bio-production. Applicants have also developed variant insoluble carrier polypeptides (e.g., ranpirnase polypeptides or TAF12 polypeptides) that exhibit increased expression in inclusion bodies when expressed in host cells. Thus, these variant ranpirnase polypeptides can be used as fusion partners for increasing the biological production of a peptide of interest operably linked to the variant ranpirnase. Without wishing to be bound by theory, the increased accumulation of the peptide of interest in inclusion bodies allows higher levels of peptide to be purified from the host cell.

As used herein, "amino acid" or "amino acid residue" refers to any naturally occurring amino acid, any non-naturally occurring amino acid, any modified amino acid (including derivatized amino acids), or any amino acid mimetic known in the art. Amino acids may be referred to by both their common three-letter abbreviations and single-letter abbreviations.

As used herein, the term "peptide" refers to any peptide structure comprising or consisting of two or more amino acids (including chemical modifications and derivatives of amino acids). In some embodiments, the peptide is a short peptide (e.g., 4 to 50 amino acids in length). In some embodiments, the peptide is an oligopeptide that is released when the polypeptide is cleaved.

As used herein, the term "purified" molecule refers to a biological or synthetic molecule that is removed from its natural environment and is isolated or separated and free of other components with which it is naturally associated.

The term "isolated" with respect to molecules, including nucleic acids, constructs, vectors, and the like, may refer to molecules that are not found in nature and/or molecules that are present in environments not found in nature. The term "isolated" may also refer to a molecule that has undergone at least one step intended to be separated or concentrated or enriched from a more complex solution or source. However, the term "isolated" is in no way intended to limit the molecules to a particular location or state. For example, an isolated nucleic acid molecule includes a nucleic acid molecule that is introduced into the genome of a cell at a location not found in nature, or that is introduced into the genome of a cell into which it has been introduced while it remains in the progeny of the cell.

The term "sequence identity" refers to the degree of similarity between two nucleic acid sequences or two amino acid sequences, expressed as the similarity between the sequences, and is otherwise referred to as sequence identity. Sequence identity is typically measured in terms of percent identity (or similarity or homology), with higher percentages being more similar the two sequences.

As used herein, "concatemer polypeptide (concatemeric polypeptide)" or "concatemer" refers to a polypeptide that includes multiple copies of a given unit (e.g., an oligopeptide) as tandem repeats. In some embodiments, the concatemer polypeptide is a homologous concatemer (each oligopeptide unit is identical). In some embodiments, the concatemer polypeptide is a heterologous concatemer (more than one different oligopeptide is present). In some embodiments, the concatemer polypeptide comprises a linker between the units.

As used herein, "active amino acid sequence," "bioactive amino acid sequence," and "active sequence" refer to an amino acid sequence capable of inducing a biological effect in a living cell and/or organism exposed to the amino acid sequence. In some embodiments, the active amino acid sequence has microbial inhibitory activity. In some embodiments, the active amino acid sequence confers a plant benefit.

As used herein, a "ranpirnase variant" is a ranpirnase polypeptide comprising one or more amino acid insertions, deletions or substitutions compared to the amino acid sequence set forth in SEQ ID No. 1.

I. method for producing oligopeptides

Some aspects of the disclosure provide methods of producing oligopeptides. In some embodiments, a method for producing an oligopeptide comprises expressing a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) operably linked to two or more oligopeptides by peptide bonds, and releasing the two or more oligopeptides from the insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) by sequence-specific chemical cleavage of the peptide bonds.

Fusion proteins

In some embodiments, a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) is operably linked to two or more oligopeptides. In some embodiments, the method comprises expressing a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) and two or more polypeptides. For example, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) operably linked to 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, 15 or more oligopeptides, 20 or more oligopeptides. In some embodiments, the fusion polypeptide comprises two or more oligopeptides having the same amino acid sequence. In some embodiments, the fusion polypeptide comprises two or more oligopeptides having different amino acid sequences. In some embodiments, the fusion polypeptide comprises two or more oligopeptides having the same amino acid sequence and two or more oligopeptides comprising different amino acid sequences.

In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) and two or more oligopeptides operably linked to the N-terminus and/or the C-terminus of the ranpirnase protein. For example, the fusion polypeptide can comprise two or more oligopeptides operably linked to each of the N-terminus and the C-terminus of an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide). In some embodiments, the fusion polypeptide comprises a total of 2, 3,4,5,6, 7, 8, 9, 10, 15, 20, 25, or more oligopeptides operably linked to the N-terminus and/or the C-terminus of the ranpirnase protein. In some embodiments, the number of oligopeptides operably linked to each end of ranpirnase may be the same or different.

In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide), as well as an oligopeptide that is not operably linked to the N-terminus of the insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) and two or more oligopeptides that are operably linked to the C-terminus. In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide), as well as an oligopeptide that is not operably linked to the N-terminus of the insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) and 4, 5, or 6 oligopeptides operably linked to the C-terminus. In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide), and two or more oligopeptides operably linked to the N-terminus of the insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) and 3 oligopeptides operably linked to the C-terminus. In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide), and 1,2, or 3 oligopeptides operably linked to the N-terminus and 3 oligopeptides operably linked to the C-terminus of the insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide). In some embodiments, the one or more oligopeptides operably linked to the N-terminus of ranpirnase may have the same amino acid sequence as the one or more oligopeptides operably linked to the C-terminus of an insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide). In some embodiments, the one or more oligopeptides operably linked to the N-terminus of ranpirnase have an amino acid sequence that is different from the one or more oligopeptides operably linked to the C-terminus of ranpirnase. In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) and two or more oligopeptides linked to the C-terminus of ranpirnase. In some embodiments, the two or more oligopeptides linked to the C-terminus of ranpirnase have the same amino acid sequence. In some embodiments, the two or more oligopeptides linked to the C-terminus of ranpirnase have different amino acid sequences. In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) and two or more oligopeptides linked to the N-terminus of ranpirnase. In some embodiments, the two or more oligopeptides linked to the N-terminus of ranpirnase have the same amino acid sequence. In some embodiments, the two or more oligopeptides linked to the N-terminus of ranpirnase have different amino acid sequences. In some cases, an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) is separated from two or more oligopeptides by peptide bonds. In some embodiments, two or more oligopeptides are themselves separated by a peptide bond. In some embodiments, the peptide bond comprises a cleavable bond.

The cleavable bond may be cleaved by an enzyme or chemical reagent. In certain embodiments, the cleavable bond is a peptide bond. Cleavage of the peptide bond can result in separation of the insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) from the oligopeptide and/or separation of the oligopeptides from each other. Any cleavable peptide bond that can be cleaved by any method or agent in the art can be used with the methods of the present disclosure. Proteolytic enzymes and their respective cleavage site specificities are well known in the art. Examples of enzymes that can be used to cleave a peptide linker include, but are not limited to, arg-C protease, asp-N endopeptidase, chymotrypsin, clostripain, enterokinase, factor Xa, glutamyl endopeptidase, granzyme B, achromobacter protease I, pepsin, proline endopeptidase, proteinase K, staphylococcal peptidase I, thermolysin, thrombin, trypsin, TEV protease, HRV3C, and Caspase family members of proteases (e.g., caspases 1-10). Chemical cleavage agents are also well known in the art and can result in cleavage of a polypeptide at a peptide bond between a particular pair of amino acids. Examples of chemical cleavage reagents include cyanogen bromide (cleavage after a methionine residue to produce a C-terminal homoserine lactone in place of methionine), N-chlorosuccinimide, iodobenzoic acid, or BNPS-skatole [2- (2-nitrophenylsulfinyl) -3-methylindole ] (each of which cleaves a tryptophan residue to produce a C-terminal tryptophan (and lactone)), dilute acid (which cleaves at an aspartyl-prolyl bond to produce a C-terminal aspartic acid and an N-terminal proline), and hydroxylamine (which cleaves at an asparagine-glycine bond to produce a C-terminal aspartic acid and an N-terminal glycine at pH 9.0). Additional chemical cleavage agents are described in Gavit, P.and Better, M., J.Biotechnol., 79:127-136 (2000), szoka et al, DNA, 5 (1): 11-20 (1986), and Walker, J.M., the Proteomics Protocols Handbook, 2005, humana Press, totowa, N.J.), respectively. In some embodiments, the peptide cleavable bond between the peptides can be cleaved by the same reagent as cleaves the peptide cleavable bond between the peptide and ranpirnase. In some embodiments, the peptide cleavable bond between the peptides can be cleaved by an agent that is different from the peptide cleavable bond between the cleavage peptide and ranpirnase.

In some embodiments, cleavage occurs at a specific amino acid sequence. In some embodiments, the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is with formic acid, acetic acid or any dilute acid solution, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is with NTCB. In some embodiments, two or more oligopeptides are operably linked to an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) through a peptide bond comprising an Asp-Pro bond, and the sequence-specific chemical cleavage is with formic acid or acetic acid. In some embodiments, the peptides are linked to each other by peptide bonds comprising Asp-Pro bonds, and the sequence-specific chemical cleavage is performed with formic acid or acetic acid.

The two or more oligopeptides may correspond to any desired amino acid sequence. In some cases, the oligopeptide may be a small peptide (e.g., 4 to 25 amino acids in length). For example, the oligopeptide may be at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 amino acids. Alternatively, the oligopeptide may be a longer peptide (e.g., up to 50 amino acids in length). The oligopeptides may be less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, or less than 20 amino acids in length. In some cases, the oligopeptides are 4 to 50, 6 to 40, 6 to 30, or 8 to 25 amino acids in length.

In some embodiments, one or more of the oligopeptides comprises the amino acid sequence of any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, one or more oligopeptides comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 12. In some embodiments, one or more oligopeptides comprise an amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 13. In some embodiments, one or more oligopeptides comprise at least one amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 14.

In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) linked to two or more micro-peptides (miPEP). miPEP is a short peptide (7-44 amino acids long) defined by one or several short open reading frames located in the pri-microRNA sequence of a specific miRNA member. Without wishing to be bound by theory, miPEP target its pri-micrornas encoding mirnas to up-regulate or down-regulate transcription. miPEP can target mirnas involved in various processes, such as mirnas involved in immunity and susceptibility to pathogens (e.g., microorganisms), organogenesis, response to stress, embryonic development, and the like. In some embodiments, each of the two or more oligopeptides comprises miPEP sequences that regulate the miRNA. In some embodiments, the two or more oligopeptides comprise miPEP sequences that regulate one or more families of mirnas. In some embodiments, each of the two or more oligopeptides comprises a miPEP sequence that modulates one or more members of a particular miRNA family. In some embodiments, each of the two or more oligopeptides comprises a miPEP sequence that modulates a plant miRNA, a fungal miRNA, or a metazoan miRNA. In some embodiments, each of the two or more oligopeptides comprises miPEP sequences that regulate plant mirnas. Exemplary plant microRNA (microRNA) families that can be regulated by miPEP include, but are not limited to, plant miRNA family miR156、miR159/319、miR160、miR162、miR164、miR166、miR167、miR168、miR169、miR171、miR172、miR390、miR393、miR394、miR295、miR396、miR397、miR398、miR408、miR403、miR437、miR444 and miR445. In some embodiments, each of the two or more oligopeptides comprises a miPEP sequence having microbial inhibitory activity.

In some embodiments, the oligopeptide comprises any other peptide of interest. In some embodiments, the oligopeptide comprises a polypeptide that is toxic to the host cell. In some embodiments, the oligopeptide comprises a therapeutic polypeptide, toxin, cytokine, peptide hormone, clotting factor, immunogenic peptide, allergen, antimicrobial peptide, ligand binding domain, or any combination thereof. In some embodiments, an immunogenic peptide is a peptide that can induce an immune response to a polypeptide comprising the peptide. In some embodiments, the one or more oligopeptides comprise peptide fragments of a larger polypeptide. For example, one or more oligopeptides may comprise a peptide fragment of a receptor, enzyme, adhesion molecule, or structural protein.

In some embodiments, the oligopeptide comprises the active amino acid sequence and a tag at the N-terminus and/or C-terminus. In some embodiments, the N-terminal and/or C-terminal residues may correspond to cleavage tags generated upon release of the oligopeptide. For example, the residue may be part of a peptide bond cleaved by chemical cleavage (e.g., acid cleavage) or enzymatic reaction. In some embodiments, the oligopeptide comprises the active amino acid sequence and N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid. In some embodiments, the active amino acid sequence has antimicrobial activity.

The fusion polypeptide can also comprise a linker sequence between two or more oligopeptides and/or between an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) and an oligopeptide. The linker sequence may be used as a spacer peptide to separate two or more oligopeptides and/or insoluble carrier polypeptides (e.g., ranpirnase polypeptides or TAF12 polypeptides) from oligopeptides. Any number of linker sequences known in the art may be used. In some embodiments, the linker is about 1 to 50 amino acids in length. In some embodiments, the linker is about 4 to 8 amino acids in length. In some embodiments, the linker sequence is a flexible amino acid sequence. In some embodiments, the flexible linker comprises glycine, serine, or threonine residues, or any combination thereof. In some embodiments, the flexible linker sequence comprises a (GGGGS) _n (SEQ ID NO: 2402) or (G) _n sequence, where n is an integer. In some embodiments, the flexible linker comprises the amino acid sequences GGG, GSGS (SEQ ID NO: 30), GSGSGGT (SEQ ID NO: 31), GGSGTG (SEQ ID NO: 27) or GTGSGTG (SEQ ID NO: 32). In some embodiments, the linker sequence is a rigid linker sequence. In some embodiments, the rigid linker sequence comprises a (EAAAK) _n (SEQ ID NO: 33) or (Xp) n _n sequence, wherein n is an integer and X is any amino acid. In some embodiments, the linker sequence comprises a flexible linker sequence and a rigid linker sequence. The linker sequence may also provide cleavable peptide bonds by incorporating a cleavable linker sequence comprising an amino acid sequence that directs sequence-specific cleavage of the fusion polypeptide. In some embodiments, the linker sequence may comprise a sequence for protease or chemical cleavage. In some embodiments, the fusion polypeptide comprises a linker sequence comprising the amino acid sequence of SEQ ID NO. 11. In some embodiments, the fusion polypeptide comprises a linker sequence comprising the amino acid sequence of SEQ ID NO. 11. In some embodiments, the fusion polypeptide comprises a linker sequence comprising the amino acid sequence of SEQ ID NO. 11. In some embodiments, the linker comprises an Asp-Pro sequence.

In some embodiments, the fusion polypeptide comprises a linker sequence separating two or more oligopeptides and/or a linker sequence separating an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) from two or more oligopeptides. In some embodiments, the linker sequence separating the two or more oligopeptides has the same sequence as the linker separating the ranpirnase sequence from the oligopeptides. In some embodiments, the linker sequence located between the oligopeptides is different from the linker sequence separating the insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) from two or more oligopeptides. In some embodiments, the linker sequence provides a cleavable peptide bond. In some embodiments, the linker sequence comprises an amino acid sequence for sequence-specific cleavage of the linker. In some embodiments, the sequence-specific cleavage is an acidic cleavage. In some embodiments, the fusion polypeptide includes a linker sequence comprising the amino acid sequence of SEQ ID NO. 11 that separates two or more oligopeptides from each other and/or separates an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) from two or more oligopeptides. In some embodiments, the fusion polypeptide includes a linker sequence comprising an Asp-Pro linker that separates two or more oligopeptides from each other and/or separates an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) from two or more oligopeptides.

In some embodiments, the fusion polypeptide further comprises one or more amino acid sequence tags. In some embodiments, the amino acid sequence tag does not affect the solubility of the ranpirnase fusion protein. In some embodiments, the amino acid sequence tag has a neutral pI. In some embodiments, the one or more amino acid sequence tags are purification tag peptides. Such tag peptides may include, but are not limited to, glutathione-S-transferase (GST), polyhistidine, maltose Binding Protein (MBP), avidin, biotin, streptavidin, histidine (His) tags (e.g., his-6X tags), V5 tags, FLAG tags, influenza Hemagglutinin (HA) tags, myc tags, VSV-G tags, thioredoxin (Trx) tags, and ligands for cellular receptors (e.g., insulin receptor ligands). In some embodiments, the one or more amino acid sequence tags are reporter tag peptide sequences. Examples of reporter tag peptide sequences include, but are not limited to, horseradish peroxidase (HRP), chloramphenicol Acetyl Transferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, and fluorescent proteins (e.g., GFP, CFP, YFP, BFP, etc.).

In some embodiments, one or more amino acid sequence tags are operably linked to the N-terminus and/or the C-terminus of an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) or two or more oligopeptides. In some embodiments, one or more amino acid sequence tags are operably linked to the N-terminus and/or the C-terminus of an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide). In some embodiments, one or more tag peptides are operably linked to the C-terminus of an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide). In some embodiments, one or more tag peptides are operably linked to the N-terminus of an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide). In some embodiments, one or more amino acid sequence tags are operably linked to the N-terminus and/or the C-terminus of two or more oligopeptides. In some embodiments, one or more tag peptides are operably linked to the N-terminus of two or more oligopeptides. In some embodiments, one or more tag peptides are operably linked to the C-terminus of two or more oligopeptides. In some embodiments, one or more amino acid sequence tags are operably linked to the C-terminus of an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) and the N-terminus of two or more oligopeptides. In some embodiments, one or more amino acid sequence tags are operably linked to the N-terminus of ranpirnase and the C-terminus of one or more oligopeptides. In some embodiments, one or more amino acid sequence tags are operably linked to the C-terminus of one oligopeptide and the N-terminus of another oligopeptide. In some embodiments, the amino acid sequence tag operably linked to an insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) is the same as the amino acid sequence tag operably linked to two or more oligopeptides. In some embodiments, the amino acid sequence tag operably linked to an insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) is different from the amino acid sequence tag operably linked to two or more oligopeptides.

Ranpirnase is an RNAse that exhibits a pH dependent change in solubility. The solubility of ranpirnase is determined by its isoelectric point (pI). The pI of a protein is the pH at which the protein does not carry a net charge. Under acidic conditions, ranpirnase is positively charged and soluble when the pH is below pI. Under neutral conditions, ranpirnase is neutrally charged and forms insoluble aggregates when the pH is around pI. These insoluble aggregates can be removed from the solution by selective precipitation. pI corresponds to the average value of the acid dissociation constants or pKa of amino acids in a particular protein. Thus, pI is higher when the protein contains more Arg (pKa = 12.48, Lys (pKa = 10.79), Tyr (pKa = 10.07), Cys (pKa = 8.35), His, (pKa = 6.04), Asp (pKa = 3.86) and Glu (pka=4.25) residues, and lower when the protein contains more nonpolar amino acid residues. Ranpirnase is a high cationic protein with pI higher than 9.5. In some embodiments, the ranpirnase has a pI of about 7.5. In some embodiments, the ranpirnase has a neutral pI. Furthermore, in some embodiments, ranpirnase lacks any aspartate-proline and asparagine-glycine sequences, which makes it resistant to common chemical cleavage strategies such as acid cleavage.

In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide comprising the amino acid sequence of SEQ ID NO. 1 operably linked to two or more oligopeptides via peptide bonds. In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 1. In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide comprising an amino acid sequence that has 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity with SEQ ID No. 1. In some embodiments, the ranpirnase polypeptide is a truncated ranpirnase polypeptide.

In some embodiments, the fusion polypeptide comprises a variant ranpirnase polypeptide operably linked to two or more oligopeptides through peptide bonds. The variant ranpirnase may be a ranpirnase having a mutation that reduces sensitivity to protein cleavage, increases yield, promotes aggregation, or promotes solubilization. For example, M23L-ranpirnase (M23L-ONC) disclosed in Pane et al (Pane, K., et al (2016) PLoS one.11 (1): e0146552, incorporated herein by reference) does not contain any internal methionine residues and is less susceptible to chemical cleavage strategies. The modification may be one or more of an amino acid substitution, deletion or insertion. Other amino acid substitutions, insertions or deletions may be introduced which increase the stability, yield or aggregation of the ranpirnase protein.

In some embodiments, the fusion polypeptide comprises a variant ranpirnase comprising one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO. 1. In some embodiments, one or more amino acid substitutions is a conservative amino acid substitution. Conservative amino acid substitutions refer to the substitution of a residue having a similar side chain. For example, the amino acid groups having aliphatic side chains are glycine, alanine, valine, leucine and isoleucine, the amino acid groups having aliphatic-hydroxyl side chains are serine and threonine, the amino acid groups having amide-containing side chains are asparagine and glutamine, the amino acid groups having aromatic side chains are phenylalanine, tyrosine and tryptophan, the amino acid groups having basic side chains are lysine, arginine and histidine, and the amino acid groups having sulfur-containing side chains are cysteine and methionine. In some embodiments, one or more amino acid substitutions is a non-conservative amino acid substitution. Non-conservative substitutions refer to substitutions between amino acids having different properties (e.g., an uncharged residue is substituted by a charged residue). In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO. 1. The one or more insertions or deletions may be of any amino acid length.

In some embodiments, the fusion protein comprises a ranpirnase polypeptide having one or more amino acid substitutions that reduce the sensitivity of the ranpirnase polypeptide to chemical cleavage as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, the fusion protein comprises a ranpirnase polypeptide having one or more deletions or insertions that reduce the sensitivity of the ranpirnase polypeptide to chemical cleavage as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1.

In some embodiments, the fusion protein comprises a ranpirnase polypeptide having one or more amino acid substitutions that increase yield or promote inclusion body formation as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, the one or more amino acid substitutions that increase yield or promote inclusion body formation include substitution of one or more cationic or polar amino acids for non-polar or neutral amino acids. In some embodiments, the ranpirnase comprises one or more amino acid substitutions, insertions, or deletions that reduce solubility. In some embodiments, the one or more amino acid substitutions result in the variant ranpirnase polypeptide having a reduced number of cationic or polar amino acids compared to the ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, one or more amino acid substitutions that increase yield or promote inclusion body formation result in a variant ranpirnase having a reduced number of Lys, arg, cys, his, asp, glu and/or Tyr compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, the fusion protein comprises a ranpirnase polypeptide having one or more amino acid substitutions that result in a ranpirnase protein having an altered pI as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, the fusion protein comprises a ranpirnase polypeptide having one or more deletions or insertions as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1, which one or more deletions or insertions increases yield or promotes inclusion body formation. In some embodiments, one or more insertions or deletions that increase yield or promote inclusion body formation result in a variant ranpirnase having a reduced number of cationic or polar amino acids compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, one or more insertions or deletions that increase yield or promote inclusion body formation result in a variant ranpirnase with a reduced number of Lys, arg, cys, his, asp, glu and/or Tyr compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, the fusion protein comprises a ranpirnase polypeptide having one or more deletions or insertions as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1, which one or more deletions or insertions increases yield or promotes inclusion body formation. In some embodiments, the fusion protein comprises a ranpirnase polypeptide having one or more deletions or insertions that result in a ranpirnase protein having an altered pI as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In any of the above embodiments, the ranpirnase polypeptide can comprise an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 1. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity with SEQ ID No. 1.

In some embodiments, the fusion protein comprises a ranpirnase polypeptide having one or more N-terminal amino acid substitutions as compared to SEQ ID No. 1. In some embodiments, the fusion protein comprises a ranpirnase polypeptide having 1, 2, 3, 4,5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, or 35N-terminal amino acid substitutions as compared to SEQ ID No. 1. In some embodiments, the fusion protein comprises a ranpirnase polypeptide having 11N-terminal amino acid substitutions as compared to SEQ ID No. 1. In some embodiments, the amino acid substitution is within the first 50N-terminal amino acids, within the first 40N-terminal amino acids, within the first 30N-terminal amino acids, within the first 20N-terminal amino acids, or within the first 11 amino acids. In some embodiments, the amino acid substitution is within the first 15N-terminal amino acids, within the first 14N-terminal amino acids, within the first 13N-terminal amino acids, within the first 12N-terminal amino acids, within the first 11N-terminal amino acids, within the first 10N-terminal amino acids, within the first 9N-terminal amino acids, within the first 8N-terminal amino acids, within the first 7N-terminal amino acids, within the first 6N-terminal amino acids, within the first 5N-terminal amino acids, within the first 4N-terminal amino acids, within the first 3N-terminal amino acids, or within the first 2N-terminal amino acids. In some embodiments, the one or more N-terminal amino acid substitutions are conservative amino acid substitutions. In some embodiments, the one or more N-terminal amino acid substitutions are non-conservative amino acid substitutions. In some embodiments, the fusion protein comprises a ranpirnase polypeptide having one or more N-terminal insertions or deletions compared to SEQ ID No. 1. In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide comprising the amino acid sequence of SEQ ID NO. 1. In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 1. In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide comprising an amino acid sequence that has 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity with SEQ ID No. 1.

In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising the amino acid sequence of any of SEQ ID NOs 2-10 or 15-22, optionally with 1,2, 3, 4, 5, or all 6C-terminal histidine residues deleted, as shown in FIG. 20A. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 2. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 3. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 4. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 5. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 6. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 7. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 7. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 8. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 9. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 10. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 15. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 16. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 17. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 18. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 19. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 20. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 21. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 22. In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide comprising at least one amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any of SEQ ID NOs 2-10 or 15-22, optionally with a deletion of 1,2, 3, 4, 5, or all 6C-terminal histidine residues, as shown in fig. 20A.

In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide having a modified N-terminus and/or C-terminus. In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide having one or more amino acid substitutions at the N-terminus and/or C-terminus as compared to SEQ ID No. 1. In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide comprising 11 amino acid substitutions at the N-terminus and/or C-terminus compared to SEQ ID No. 1. In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide comprising 11 amino acid substitutions at the N-terminus compared to SEQ ID No. 1.

In some embodiments, the fusion polypeptide comprises a TAF12 polypeptide comprising the amino acid sequence of SEQ ID NO. 23 operably linked to two or more oligopeptides via peptide bonds. In some embodiments, the fusion polypeptide comprises a TAF12 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO. 23. In some embodiments, the fusion polypeptide comprises a TAF12 polypeptide comprising an amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to SEQ ID NO. 23. In some embodiments, the TAF12 polypeptide is a truncated TAF12 polypeptide.

In some embodiments, the fusion polypeptide comprises a variant TAF12 polypeptide operably linked to two or more oligopeptides by peptide bonds. The variant TAF12 may be TAF12 having mutations that reduce sensitivity to protein cleavage, increase yield, promote aggregation, or promote lysis. The modification may be one or more of an amino acid substitution, deletion or insertion. Other amino acid substitutions, insertions or deletions may be introduced which increase the stability, yield or aggregation of the TAF12 protein.

In some embodiments, the fusion polypeptide comprises a variant TAF12 comprising one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO. 23. In some embodiments, one or more amino acid substitutions is a conservative amino acid substitution. Conservative amino acid substitutions refer to the substitution of a residue having a similar side chain. For example, the amino acid groups having aliphatic side chains are glycine, alanine, valine, leucine and isoleucine, the amino acid groups having aliphatic-hydroxyl side chains are serine and threonine, the amino acid groups having amide-containing side chains are asparagine and glutamine, the amino acid groups having aromatic side chains are phenylalanine, tyrosine and tryptophan, the amino acid groups having basic side chains are lysine, arginine and histidine, and the amino acid groups having sulfur-containing side chains are cysteine and methionine. In some embodiments, one or more amino acid substitutions is a non-conservative amino acid substitution. Non-conservative substitutions refer to substitutions between amino acids having different properties (e.g., an uncharged residue is substituted for a charged residue). In some embodiments, the fusion protein comprises a TAF12 polypeptide comprising one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO. 23. The one or more insertions or deletions may be of any amino acid length.

In some embodiments, the fusion protein comprises a TAF12 polypeptide having one or more amino acid substitutions that reduce the sensitivity of the TAF12 polypeptide to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23. In some embodiments, the fusion protein comprises a TAF12 polypeptide having one or more deletions or insertions that reduce the sensitivity of the TAF12 polypeptide to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23.

In some embodiments, the fusion protein comprises a TAF12 polypeptide having one or more amino acid substitutions that increase yield or promote inclusion body formation as compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23. In some embodiments, the one or more amino acid substitutions that increase yield or promote inclusion body formation include substitution of one or more cationic or polar amino acids for non-polar or neutral amino acids. In some embodiments, TAF12 comprises one or more amino acid substitutions, insertions, or deletions that reduce solubility. In some embodiments, the one or more amino acid substitutions results in a variant TAF12 polypeptide having a reduced number of cationic or polar amino acids compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23. In some embodiments, one or more amino acid substitutions that increase yield or promote inclusion body formation result in a variant TAF12 having a reduced number of Lys, arg, cys, his, asp, glu and/or Tyr compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23. In some embodiments, the fusion protein comprises a TAF12 polypeptide having one or more amino acid substitutions that result in a TAF12 protein having an altered pI compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO. 23. In some embodiments, the fusion protein comprises a TAF12 polypeptide having one or more deletions or insertions compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23, which one or more deletions or insertions increases yield or promotes inclusion body formation. In some embodiments, one or more insertions or deletions that increase yield or promote inclusion body formation result in a variant TAF12 having a reduced number of cationic or polar amino acids compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23. In some embodiments, one or more insertions or deletions that increase yield or promote inclusion body formation result in a variant TAF12 having a reduced number of Lys, arg, cys, his, asp, glu and/or Tyr compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23. In some embodiments, the fusion protein comprises a TAF12 polypeptide having one or more deletions or insertions compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23, one or more deletions or insertions increasing yield or promoting inclusion body formation. In some embodiments, the fusion protein comprises a TAF12 polypeptide having one or more deletions or insertions that result in a TAF12 protein having an altered pI as compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23. In any of the above embodiments, the TAF12 polypeptide may comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO. 23. In some embodiments, the TAF12 polypeptide comprises an amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to SEQ ID NO. 23.

In some embodiments, the fusion protein comprises a TAF12 polypeptide having one or more N-terminal amino acid substitutions as compared to SEQ ID NO. 23. In some embodiments, the fusion protein comprises a TAF12 polypeptide having 1,2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, or 35N-terminal amino acid substitutions compared to SEQ ID No. 23. In some embodiments, the fusion protein comprises a TAF12 polypeptide having 11N-terminal amino acid substitutions compared to SEQ ID NO. 23. In some embodiments, the amino acid substitution is within the first 50N-terminal amino acids, within the first 40N-terminal amino acids, within the first 30N-terminal amino acids, within the first 20N-terminal amino acids, or within the first 11 amino acids. In some embodiments, the amino acid substitution is within the first 15N-terminal amino acids, within the first 14N-terminal amino acids, within the first 13N-terminal amino acids, within the first 12N-terminal amino acids, within the first 11N-terminal amino acids, within the first 10N-terminal amino acids, within the first 9N-terminal amino acids, within the first 8N-terminal amino acids, within the first 7N-terminal amino acids, within the first 6N-terminal amino acids, within the first 5N-terminal amino acids, within the first 4N-terminal amino acids, within the first 3N-terminal amino acids, or within the first 2N-terminal amino acids. In some embodiments, the one or more N-terminal amino acid substitutions are conservative amino acid substitutions. In some embodiments, the one or more N-terminal amino acid substitutions are non-conservative amino acid substitutions. In some embodiments, the fusion protein comprises a TAF12 polypeptide having one or more N-terminal insertions or deletions compared to SEQ ID NO. 23. In some embodiments, the fusion polypeptide comprises a TAF12 polypeptide comprising the amino acid sequence of SEQ ID NO. 23. In some embodiments, the fusion polypeptide comprises a TAF12 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO. 23. In some embodiments, the fusion polypeptide comprises a TAF12 polypeptide comprising an amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to SEQ ID NO. 23.

Peptide release

In some embodiments, the method of producing an oligopeptide comprises releasing two or more oligopeptides from an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) by sequence-specific cleavage of a peptide bond. In some embodiments, sequence-specific cleavage of the peptide bond is performed by acidic cleavage. In some embodiments, the acidic cleavage is performed by incubation with acetic acid or formic acid. In some embodiments, the incubation is performed at an elevated temperature, such as above room temperature.

In some embodiments, the oligopeptides are operably linked by peptide bonds and released from each other when released from ranpirnase. In some embodiments, the oligopeptides are operably linked by different peptide bonds and released from each other by sequence-specific chemical cleavage of the different peptide bonds after the oligopeptides are released from the ranpirnase.

In some embodiments, the step of releasing two or more oligopeptides from an insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) is performed in the presence of a chaotropic agent. Chaotropic agents are molecules capable of breaking the hydrogen bonding network between water molecules. Theoretically, the addition of chaotropic agents can reduce the structural order of proteins, thereby promoting protein unfolding. Examples of chaotropic agents include, but are not limited to, guanidine hydrochloride (guanidine hydrochloride, gdnchi), sodium thiocyanate, n-butanol, ethanol, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, sodium dodecyl sulfate, and urea. Chaotropic agents can also be detergents that disrupt non-covalent intermolecular bonds within proteins, allowing the amino acid chains to assume a substantially random conformation.

In some embodiments, the step of releasing two or more oligopeptides from an insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) is performed in the absence of a chaotropic agent. In protein purification schemes, the addition of chaotropic agents may not always be required. For example, adding a chaotropic agent may require additional steps or conditions to be added to a regimen. These additional steps may increase the amount of time, reagents and costs of the purification scheme or may negatively impact protein yield or purity.

The fusion polypeptide may be expressed in any suitable cell. The fusion polypeptide may be expressed in bacterial, yeast, plant or animal cells. In some embodiments, the fusion polypeptide is expressed in a single cell organism. In some embodiments, the fusion polypeptide is expressed in a microbial cell. Any known cell or cell culture that produces inclusion bodies is suitable for expressing the fusion peptide. For example, the fusion polypeptide may be expressed in strains of E.coli and Vibrio natriuretic. Suitable yeast cells include, but are not limited to, pichia pastoris strains. The bacterial strain may be a strain lacking Lon and ompT protease functions (e.g., a BL21 escherichia coli strain). In some embodiments, the fusion polypeptide is expressed in bacteria in a bacterial cell culture. In some embodiments, the bacterium is a strain of escherichia coli.

Expression of the polypeptide may be driven by any suitable promoter known in the art. The promoter may be a constitutive promoter or an inducible promoter. Inducible promoters refer to those regulated promoters that can be turned on by an external stimulus (e.g., chemical, nutritional stress, or heat). For example, the lac promoter may be induced by using lactose or IPTG (isopropylthio- β -D-galactoside). Alternatively, the promoter may be a constitutive promoter that directs expression of the fusion polypeptide in a particular organism, cell or tissue. The strength of the promoter, whether inducible or constitutive, may vary. For example, a promoter may be a high expression promoter or a strong promoter that results in overexpression of a given gene. In some embodiments, the fusion polypeptide is expressed from a strong promoter. In some embodiments, the promoter drives expression of the fusion protein in the aggregate.

In some embodiments, the method further comprises measuring the yield of the released oligopeptide. Protein yield may be measured by any known method for measuring peptide yield. For example, peptide yield may be measured in terms of the mass of peptide obtained from a liter of cell culture or g/L. In some embodiments, the oligopeptide is produced in a yield of at least 10 mg/L of bacterial culture, at least 20 mg/L of bacterial culture, at least 30 mg/L of bacterial culture, at least 40 mg/L of bacterial culture, at least 50 mg/L of bacterial culture. In some embodiments, the oligopeptide is produced in a yield of 10 mg/L to 200 mg/L bacterial culture, 20 mg/L to 150 mg/L bacterial culture, 30 mg/L to 120 mg/L bacterial culture, 40 mg/L to 120 mg/L bacterial culture, 50 mg/L to 120 g/L bacterial culture, 50 g/L to 100 g/L bacterial culture, 60 g/L to 90 g/L bacterial culture, 70 g/L to 110 g/L bacterial culture, or 40 g/L to 80 g/L bacterial culture. In some embodiments, the yield of expressed fusion polypeptide is measured after harvesting the cells at stationary phase.

In some embodiments, the method may comprise expressing the fusion polypeptide in a cell culture in a bioreactor. The use of a bioreactor may result in a higher yield of the released oligopeptides. Large scale cell growth and fusion polypeptide expression can utilize a wide range of simple or complex carbohydrates, organic acids or alcohols, and saturated hydrocarbons such as methane. Expression of the gene encoding the fusion protein may be regulated, repressed or inhibited by specific growth conditions, which may include the form and amount of nitrogen, phosphorus, sulfur, oxygen, carbon or any trace micronutrient, including small inorganic ions. Furthermore, modulation may be achieved by the presence or absence of specific regulatory molecules added to the culture and not normally considered a nutrient or energy source.

Method for releasing oligopeptides from insoluble carrier polypeptides forming inclusion bodies

Some aspects of the disclosure provide methods of releasing an oligopeptide fused to an insoluble carrier polypeptide that forms inclusion bodies in a cell.

Inclusion bodies are intracellular amorphous deposits comprising collectins found in the cytoplasm or periplasm of cells. The oligopeptides of interest, which are typically soluble in cells and/or cell lysates, may be linked to an insoluble carrier polypeptide, which forms inclusion bodies to promote the formation of insoluble aggregates comprising the oligopeptides. Oligopeptides are captured in inclusion bodies, making cellular proteases inaccessible and can accumulate in large quantities. Examples of insoluble carrier polypeptides that form inclusion bodies include, but are not limited to, trpΔLE polypeptides (Derynck R et al (1984)), ketosteroid isomerase polypeptides (Kuliopulos et al (1994)), beta-galactosidase polypeptides (Schellenberger V et al (1993)), pagP polypeptides (Hwang PM et al (2012)), EDDIE (Achmuller C et al (2007)), cleavable self-aggregating tags INTEIN-ELK16 (Zhao Q et al (2016)), a, GFIL8 (Wang X. Et al (2015)), paP3.30 polypeptide (Rao XC et al (2004)), TAF12-HFD (Vidovic V et al (2012)) and F4 fragment of PurF (Lee JH et al (2000)). Other exemplary insoluble carrier polypeptides that form inclusion bodies include modified versions of E.coli maltose binding protein (Betton and Hofnug, J.biol. Chem.271:8046-8052 (1996)), E.coli RNAse II polypeptide (Coburn AND MACKIE, J.biol chem.271:1048-1053 (1996)), E.coli alkaline phosphatase polypeptide (Derman and Beckwith,J Bacteriol177:3764-3770 (1995); Georgiou et al., Appl.Env.Microbial.52:1157-1161 (1986))、 E.coli phospholipase A polypeptide (Dekker et al, eur. J. Biochem.232:214-219 (1995)), and combinations thereof, Coli beta-lactamase polypeptide (Rinas and Bailey, Appl.Env.Microbiol.59:561-566 (1993); Georgiou et al., Appl.Env.Microbiol.52:1157-1161 (1986))、 Salmonella typhimurium MalK protein (SCHNEIDER ET al., prot. Exp. Purif.6:10-14 (1995)), clostridium thermocellum endoglucanase D polypeptide (Tokatlidis et al., FEBS Lett.282:205-208 (1991)), bacillus thuringiensis subspecies aizawai IPL7 insecticidal protein (Oeda et al., J. Bacteriol.171:3568-3571 (1989)), and a pharmaceutical composition comprising the same, Human tissue Proprotein (procathepsin) B polypeptide (Kuhelj et al, eur.J. biochem.229:533-539 (1995)), porcine interferon-gamma polypeptide (Vandenbroeck et al, eur.J. biochem.215:481-486 (1993)), T5 DNA polymerase polypeptide (Chatterjee et al, gene 97:13-19 (1991)), E.coli thioredoxin (Hoog et al, bioSci.Rep.4:917-923 (1984)). in some embodiments, the insoluble carrier polypeptide is a ranpirnase polypeptide or a TAF12 polypeptide.

In some embodiments, a method of releasing an oligopeptide fused to an insoluble carrier polypeptide that forms inclusion bodies in a cell comprises expressing a fusion polypeptide comprising an oligopeptide operably linked to an insoluble carrier polypeptide that forms inclusion bodies in a cell, and releasing two or more oligopeptides from the insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) by sequence-specific chemical cleavage of peptide bonds.

In some embodiments, the method comprises purifying the inclusion bodies. Inclusion bodies can be purified by lysing cells to extract inclusion bodies. Any number of methods may be used to lyse the cells, including mechanical and/or chemical lysis. Any mechanical method known in the art for purifying inclusion bodies from cell lysates may be used, including centrifugation, filtration, sonication, french press (FRENCH PRESS), shearing, and any combination thereof.

Inclusion bodies can also be isolated by dissolving aggregates using micelles or inverse micelles as described, for example, in Vinogradov, et al (2003) al biochem.15; 320 (2): 234-8. In some embodiments, the purified inclusion bodies comprise centrifugation inclusion bodies. In some embodiments, centrifuging the inclusion bodies results in separation of the inclusion bodies from the cell lysate. In some embodiments, the inclusion bodies are solubilized in a solution comprising a chemical cleavage agent after purification. In some embodiments, the inclusion bodies are solubilized in a solution that does not contain a chaotropic agent after purification. In some embodiments, the method comprises purifying inclusion bodies formed from a fusion protein comprising ranpirnase fused to two or more oligopeptides. In some embodiments, the fusion protein is expressed in bacteria or yeast. In some embodiments, the fusion protein comprises a cleavable Asp-Pro bond. In some embodiments, the oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the ranpirnase is a variant ranpirnase.

In some embodiments, the method of purifying an inclusion body comprises expressing the fusion protein in a cell, purifying the inclusion body, and incubating the inclusion body with acetic acid, further comprising incubating the inclusion body with acetic acid. In some embodiments, the inclusion bodies are incubated with acetic acid at a higher temperature. In some embodiments, the temperature is greater than 50 ℃, greater than 60 ℃, greater than 70 ℃, greater than 80 ℃, or greater than 90 ℃. In some embodiments, the temperature is less than 100 ℃, less than 95 ℃, less than 90 ℃, or less than 85 ℃. In some embodiments, the temperature is between 50 ℃ and 60 ℃, between 60 ℃ and 70 ℃, between 70 ℃ and 80 ℃, between 80 ℃ and 90 ℃, between 50 ℃ and 100 ℃, or between 90 ℃ and 100 ℃. In some embodiments, the method comprises expressing a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) operably linked to two or more polypeptides by peptide bonds. In some embodiments, the peptide bond is capable of being sequence-specific chemical cleavage. In some embodiments, two or more oligopeptides are operably linked to an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) through a peptide bond comprising an Asp-Pro bond, and the sequence-specific chemical cleavage is with formic acid or acetic acid.

In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50 ℃ for at least one hour. The acetic acid concentration may be at least 2 wt%, at least 3 wt%, at least 4 wt%, or at least 5 wt%. In some embodiments, the concentration of acetic acid is less than 50 wt.%, less than 45 wt.%, less than 40 wt.%, less than 35 wt.%, or less than 30 wt.%. In some embodiments, the concentration of acetic acid is less than 50 wt.%, less than 45 wt.%, less than 40 wt.%, less than 35 wt.%, or less than 30 wt.%. In some embodiments, the concentration of acetic acid is less than 50 wt.%, less than 45 wt.%, less than 40 wt.%, less than 35 wt.%, or less than 30 wt.%. In some embodiments, the concentration of acetic acid is less than 50 wt.%, less than 45 wt.%, less than 40 wt.%, less than 35 wt.%, or less than 30 wt.%. In some embodiments, the acetic acid concentration is between 10 and 20 wt%, between 20 and 30 wt%, between 30 and 40 wt%, or between 40 and 50 wt%. In some embodiments, the inclusion bodies are incubated in acetic acid for 1-24 hours. In some embodiments, the inclusion bodies are incubated in acetic acid for 2 to 4 hours, 4 to 6 hours, 6 to 8 hours, 8 to 10 hours, 10 to 14 hours, 14 to 18 hours, 18 to 20 hours, or 20 to 24 hours. In some embodiments, the inclusion bodies are incubated in acetic acid for about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, or about 24 hours. In some embodiments, the inclusion bodies are in acetic acid for Wen Yoduo hours. In some embodiments, the method comprises expressing a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) operably linked to two or more polypeptides by peptide bonds. In some embodiments, the peptide bond is capable of being sequence-specific chemical cleavage. In some embodiments, two or more oligopeptides are operably linked to an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) through a peptide bond comprising an Asp-Pro bond, and the sequence-specific chemical cleavage is with formic acid or acetic acid.

In some embodiments, the method of purifying an inclusion body comprises expressing a fusion protein in a cell, purifying the inclusion body, and incubating the inclusion body with acetic acid, further comprising releasing two or more oligopeptides from an insoluble carrier polypeptide that forms the inclusion body in the cell by sequence-specific chemical cleavage. In some embodiments, the acid is used to release two or more oligopeptides from an insoluble carrier polypeptide that forms inclusion bodies in the cell. In some embodiments, the acid is acetic acid or formic acid. In some embodiments, two or more oligopeptides are released from an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) by sequence-specific acetic acid cleavage of peptide bonds. In some embodiments, cleavage of the fusion polypeptide by acetic acid results in release of the insoluble carrier polypeptide and the two or more polypeptides, and/or release of the two or more oligopeptides from each other, which form inclusion bodies in the cell. In some embodiments, the release of two or more oligopeptides from insoluble carrier polypeptides that form inclusion bodies in the cell occurs after solubilization of the inclusion bodies. In some embodiments, the release of two or more oligopeptides from insoluble carrier polypeptides that form inclusion bodies in the cells occurs during incubation of the inclusion bodies in acetic acid. In some embodiments, the release of the peptide is incomplete. For example, in some embodiments, the dipeptide or tripeptide remains after incubation with acetic acid or formic acid.

In some embodiments, the method further comprises separating the released oligopeptide from the insoluble carrier polypeptide that forms inclusion bodies in the cell. Following the release step, the oligopeptide may be separated and/or isolated from the insoluble carrier polypeptide forming inclusion bodies based on the different solubilities of the components. Parameters such as pH, salt concentration and temperature can be adjusted to facilitate separation of the oligopeptide from insoluble carrier polypeptides forming inclusion bodies. The released oligopeptides may be further purified using any purification technique known in the art, such as using, for example, ion exchange, gel purification techniques, differential centrifugation, and column chromatography.

In some embodiments, the method of purifying an inclusion body comprises expressing a fusion protein in a cell, purifying the inclusion body, and incubating the inclusion body with acetic acid, wherein the fusion polypeptide comprises expressing the fusion polypeptide under conditions that promote inclusion body formation. In some embodiments, the step of expressing the fusion polypeptide is performed at neutral pH. In some embodiments, expression of the fusion polypeptide at neutral pH results in the formation of inclusion bodies. In some embodiments, the step of expressing the fusion polypeptide is performed at a higher temperature. In some embodiments, expressing the fusion polypeptide at a higher temperature results in the formation of inclusion bodies. In some embodiments, expressing the fusion polypeptide at a high level results in the formation of inclusion bodies. In some embodiments, the step of expressing the fusion polypeptide is performed using cells that are prone to form inclusion bodies. In some embodiments, expressing the fusion polypeptide in a cell that is susceptible to inclusion body formation results in inclusion body formation.

In some embodiments, provided herein are methods of purifying inclusion bodies comprising a fusion protein, wherein the fusion protein comprises an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) operably linked to two or more oligopeptides. In some embodiments, the method comprises expressing a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) operably linked to two or more polypeptides by peptide bonds. In some embodiments, the peptide bond is capable of being sequence-specific chemical cleavage. In some embodiments, two or more oligopeptides are operably linked to an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) through a peptide bond comprising an Asp-Pro bond, and the sequence-specific chemical cleavage is with formic acid or acetic acid.

In some embodiments, the method of purifying inclusion bodies comprising a fusion protein comprises expressing a fusion polypeptide comprising two or more, 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, 15 or more, or 20 or more oligopeptides operably linked to an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide). In some embodiments, the method comprises expressing a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) and two or more oligopeptides operably linked to the N-terminus or C-terminus of the ranpirnase protein. In some embodiments, the method comprises expressing a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) and two or more oligopeptides linked to the C-terminus of the ranpirnase. In some embodiments, the two or more oligopeptides linked to the C-terminus of ranpirnase have the same amino acid sequence. In some embodiments, the two or more oligopeptides linked to the C-terminus of ranpirnase have different amino acid sequences. In some embodiments, the method comprises expressing a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) and two or more oligopeptides linked to the N-terminus of the ranpirnase. In some embodiments, the two or more oligopeptides linked to the N-terminus of ranpirnase have the same amino acid sequence. In some embodiments, the two or more oligopeptides linked to the N-terminus of ranpirnase have different amino acid sequences. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the ranpirnase is a ranpirnase mutant. In some embodiments, the ranpirnase mutant comprises one or more amino acid substitutions, insertions, or deletions in the 15N-terminal amino acids.

In some embodiments, the method of purifying an inclusion body comprises expressing a fusion protein in a cell, purifying the inclusion body, and incubating the inclusion body with acetic acid at a temperature of, for example, at least 50 ℃ for at least one hour, wherein the fusion polypeptide comprises two or more oligopeptides less than 25 amino acids in length. In some embodiments, the two or more oligopeptides are at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 amino acids in length. In some embodiments, two or more oligopeptides are up to 50 amino acids in length. In some embodiments, two or more oligopeptides are less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, or less than 20 amino acids in length. In some embodiments, the two or more oligopeptides are 4 to 50, 6 to 40, 6 to 30, or 8 to 25 amino acids in length. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50 ℃ for at least one hour.

In some embodiments, the method of purifying an inclusion body comprises expressing a fusion protein in a cell, purifying the inclusion body, and incubating the inclusion body with ethylene, wherein the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) linked to two or more micro-peptides (miPEP). In some embodiments, the two or more oligopeptides comprise miPEP sequences that regulate miRNA. In some embodiments, the two or more oligopeptides comprise miPEP sequences that regulate two or more families of mirnas. In some embodiments, the two or more oligopeptides comprise miPEP sequences that regulate plant mirnas. In some embodiments, the two or more oligopeptides comprise miPEP sequences ：miR156、miR159/319、miR160、miR162、miR164、miR166、miR167、miR168、miR169、miR171、miR172、miR390、miR393、miR394、miR295、miR396、miR397、miR398、miR408、miR403、miR437、miR444 and miR445 family that regulate a plant miRNA selected from the group consisting of. In some embodiments, one or more oligopeptides comprise miPEP sequences having microbial inhibitory activity. In some embodiments, the ranpirnase is a variant ranpirnase. In some embodiments, the two or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the fusion polypeptide is expressed in a yeast or bacterial cell. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50 ℃ for at least one hour.

In some embodiments, the method of purifying an inclusion body comprises expressing a fusion protein in a cell, purifying the inclusion body, and incubating the inclusion body with acetic acid, wherein the fusion polypeptide further comprises a linker sequence between two or more oligopeptides and/or between an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) and the oligopeptides. In some embodiments, the linker sequence is capable of being cleaved by sequence-specific chemical cleavage. In some embodiments, the linker sequence separates two or more oligopeptides and/or the linker sequence separates an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) from two or more oligopeptides. In some embodiments, the linker sequence separating the two or more oligopeptides has the same or different sequence as the linker sequence separating the insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) from the two or more oligopeptides. In some embodiments, the methods comprise expressing a fusion polypeptide comprising a linker sequence comprising the amino acid sequence of SEQ ID NO. 11 that separates two or more oligopeptides from each other and/or separates an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) from two or more oligopeptides. In some embodiments, the method comprises expressing a fusion polypeptide comprising a linker sequence comprising an Asp-Pro linker that separates two or more oligopeptides from each other and/or separates an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) from two or more oligopeptides. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50 ℃ for at least one hour.

In some embodiments, the method of purifying an inclusion body comprises expressing a fusion protein in a cell, purifying the inclusion body, and incubating the inclusion body with acetic acid, wherein the fusion polypeptide comprises a ranpirnase polypeptide comprising the amino acid sequence of SEQ ID NO. 1 operably linked to two or more oligopeptides through peptide bonds. In some embodiments, the method comprises expressing a fusion polypeptide comprising a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID No. 1. In some embodiments, the method comprises expressing a fusion polypeptide comprising a ranpirnase polypeptide comprising an amino acid sequence that has 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to SEQ ID No. 1. In some embodiments, the method comprises expressing a fusion polypeptide comprising a variant ranpirnase comprising one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO. 1. In some embodiments, the method comprises expressing a fusion polypeptide comprising a ranpirnase polypeptide comprising one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO. 1. The one or more insertions or deletions may be of any amino acid length. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50 ℃ for at least one hour.

In some embodiments, the method of purifying an inclusion body comprises expressing a fusion protein in a cell, purifying the inclusion body, and incubating the inclusion body with acetic acid, wherein the fusion polypeptide comprises a ranpirnase polypeptide having one or more amino acid substitutions that reduce the sensitivity of the ranpirnase polypeptide to chemical cleavage compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, the method comprises expressing a fusion polypeptide comprising a ranpirnase polypeptide having one or more deletions or insertions that reduce the sensitivity of the ranpirnase polypeptide to chemical cleavage as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50 ℃ for at least one hour.

In some embodiments, the method of purifying an inclusion body comprises expressing a fusion protein in a cell, purifying the inclusion body, and incubating the inclusion body with acetic acid, wherein the fusion polypeptide comprises a ranpirnase polypeptide having a modified N-terminus and/or C-terminus. In some embodiments, the method comprises expressing a fusion polypeptide comprising a ranpirnase polypeptide having one or more amino acid substitutions at the N-terminus and/or C-terminus as compared to SEQ ID No. 1. In some embodiments, the method comprises expressing a fusion polypeptide comprising a ranpirnase polypeptide comprising 11 amino acid substitutions at the N-terminus and/or C-terminus compared to SEQ ID No. 1. In some embodiments, the method comprises expressing a fusion polypeptide comprising a ranpirnase polypeptide comprising 11 amino acid substitutions at the N-terminus compared to SEQ ID No. 1. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50 ℃ for at least one hour.

In some embodiments, the method of purifying an inclusion body comprises expressing a fusion protein in a cell, purifying the inclusion body, and incubating the inclusion body with acetic acid, wherein the fusion polypeptide comprises a TAF12 polypeptide comprising the amino acid sequence of SEQ ID No. 23 operably linked to two or more oligopeptides through peptide bonds. In some embodiments, the method comprises expressing a fusion polypeptide comprising a TAF12 polypeptide, wherein the TAF12 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO. 23. In some embodiments, the method comprises expressing a fusion polypeptide comprising a TAF12 polypeptide, the TAF12 polypeptide comprising an amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to SEQ ID NO. 23. In some embodiments, the method comprises expressing a fusion polypeptide comprising a variant TAF12 comprising one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO. 23. In some embodiments, the method comprises expressing a fusion polypeptide comprising a TAF12 polypeptide comprising one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO. 23. The one or more insertions or deletions may be of any amino acid length. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50 ℃ for at least one hour.

In some embodiments, the method of purifying an inclusion body comprises expressing a fusion protein in a cell, purifying the inclusion body, and incubating the inclusion body with acetic acid, wherein the fusion polypeptide comprises a TAF12 polypeptide having one or more amino acid substitutions that reduce the sensitivity of the TAF12 polypeptide to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23. In some embodiments, the method comprises expressing a fusion polypeptide comprising a TAF12 polypeptide having one or more deletions or insertions that reduce the sensitivity of the TAF12 polypeptide to chemical cleavage as compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50 ℃ for at least one hour.

In some embodiments, the method of purifying an inclusion body comprises expressing a fusion protein in a cell, purifying the inclusion body, and incubating the inclusion body with acetic acid, wherein the fusion polypeptide comprises a TAF12 polypeptide having a modified N-terminus and/or C-terminus. In some embodiments, the method comprises expressing a fusion polypeptide comprising a TAF12 polypeptide having one or more amino acid substitutions at the N-terminus and/or C-terminus as compared to SEQ ID NO. 23. In some embodiments, the method comprises expressing a fusion polypeptide comprising a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus and/or C-terminus as compared to SEQ ID NO. 23. In some embodiments, the method comprises expressing a fusion polypeptide comprising a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus as compared to SEQ ID NO. 23. In some embodiments, the inclusion bodies are solubilized in the absence of a chaotropic agent. In some embodiments, the inclusion bodies are incubated with acetic acid at a temperature greater than 50 ℃ for at least one hour.

Methods of purifying inclusion bodies comprising fusion proteins.

Some aspects of the disclosure provide methods of purifying inclusion bodies comprising fusion proteins. In some embodiments, the method comprises expressing a fusion protein comprising an oligopeptide operably linked to a protein that forms inclusion bodies (e.g., ranpirnase), lysing the cells to form a lysate, centrifuging the cell lysate to form a pellet, washing the pellet in a surfactant buffer, washing the pellet in a salt buffer, and washing the pellet in water. In some embodiments, the method comprises purifying inclusion bodies formed from fusion proteins comprising ranpirnase fused to two or more oligopeptides. In some embodiments, the fusion protein is expressed in bacteria or yeast. In some embodiments, the fusion protein comprises a cleavable Asp-Pro bond. In some embodiments, the oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the ranpirnase is a ranpirnase mutant.

In some embodiments, the method of purifying inclusion bodies comprising a fusion polypeptide comprises lysing the cells to form a lysate. Cell lysis may be performed by any technique known to those skilled in the art. In some embodiments, cell lysis is performed by enzymatic lysis. Methods of enzymatic cleavage include, but are not limited to, enzymatic cleavage using a cleaving enzyme such as lysozyme, lysostaphin, mutanolysin, or any other cell wall disrupting enzyme. In some embodiments, cell lysis is performed by mechanical methods. Methods of mechanical lysis include, but are not limited to, physical shearing, such as with glass beads, sonication, french press (FRENCH PRESS), sonication, incubation with hypotonic solutions, or high pressure. In some embodiments, the cell lysate is a yeast or bacterial cell lysate. In some embodiments, the fusion protein comprises ranpirnase fused to two or more oligopeptides. In some embodiments, the oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the ranpirnase is a variant ranpirnase.

In some embodiments, the method of purifying inclusion bodies comprising the fusion protein comprises centrifuging the cell lysate to form a pellet. In some embodiments, centrifugation is performed at a speed of about 500xg, about 1,000 xg, about 5,000 xg, about 10,000 xg, about 15,000 xg, about 20,000 xg, or about 25,000 xg. In some embodiments, centrifugation is performed at a speed of 500xg to 1,000 xg, 1,000 xg to 5,000 xg, 5,000 xg to 10,000 xg, 10,000 xg to 15,000 xg, 15,000 xg to 20,000 xg, or 20,000 xg to 25,000 xg. In some embodiments, centrifugation is performed at a speed of about 10,000 xg. In some embodiments, centrifugation is performed at a temperature of about 4 ℃ to 10 ℃. In some embodiments, centrifugation is performed at room temperature. In some embodiments, centrifugation is performed for about 5 to 60 minutes. In some embodiments, centrifugation is performed for about 5 to 10 minutes, about 10 to 15 minutes, about 15 to 30 minutes, about 30 to 45 minutes, or about 45 to 60 minutes. In some embodiments, centrifugation is performed for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, or about 60 minutes. In some embodiments, centrifugation is performed for more than 60 minutes. In some embodiments, centrifuging the cell lysate produces a pellet comprising inclusion bodies. In some embodiments, centrifuging the cell lysate produces a pellet that is free of cell debris. In some embodiments, the cell lysate is a yeast or bacterial cell lysate. In some embodiments, the fusion protein comprises ranpirnase fused to two or more oligopeptides. In some embodiments, the oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the ranpirnase is a variant ranpirnase.

In some embodiments, the method of purifying inclusion bodies comprising a fusion protein comprises washing the precipitate in a surfactant buffer. Surfactants are surface-active compounds that reduce the surface tension of water and include nonionic (including but not limited to t-octylphenoxy polyethoxy-ethanol and polyoxyethylene sorbitan), anionic (e.g., sodium lauryl sulfate) and cationic (e.g., cetylpyridinium chloride (cetylpyridinium chloride)) and amphoteric agents. Suitable surfactants include, but are not limited to deoxycholate, sodium octyl sulfate, sodium tetradecyl sulfate, polyoxyethylene ether, sodium cholate, octyl thiopyranoside, n-octyl glucopyranoside, alkyl trimethylammonium bromide, alkyl trimethylammonium chloride, sodium bis (2-ethylhexyl) sulfosuccinate. In some embodiments, the surfactant buffer comprises a nonionic surfactant. In some embodiments, the surfactant is Triton X-100. In some embodiments, the precipitate is washed at least once, at least twice, or at least three times with a surfactant. In some embodiments, the precipitate is washed with surfactant more than three times. In some embodiments, the fusion protein comprises an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) operably linked to two or more oligopeptides via Asp-Pro bonds. In some embodiments, the oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the ranpirnase is a variant ranpirnase.

In some embodiments, the purified inclusion bodies are incubated at about 95 ℃ after washing with the surfactant. In some embodiments, the incubation denatures the protein in the inclusion bodies. In some embodiments, the incubation is performed for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 5 minutes, at least 10 minutes, or at least 20 minutes. In some embodiments, the incubation is performed for 1-20 minutes. In some embodiments, the incubation is performed between about 90 ℃ to about 100 ℃.

In some embodiments, the method of purifying inclusion bodies comprising a fusion protein comprises washing the precipitate in a salt-containing buffer. In some embodiments, the buffer has a higher salt level. Such buffers facilitate depolymerization and/or refolding of the mixture and maintenance of the desired pH value or pH range. Inorganic salt buffers (e.g., other buffers such as phosphate, carbonate, sodium, etc.) and organic salt buffers (e.g., other buffers such as citrate, tris, MOPS, MES, HEPES, etc.) are well known in the art. In some embodiments, the salt buffer comprises NaCl. In some embodiments, the salt buffer comprises at least 0.5M NaCl. In some embodiments, the salt buffer is at least 0.6M NaCl, at least 0.7M NaCl, at least 0.75M NaCl, or at least 1M NaCl. In some embodiments, the salt buffer is 0.1M to 0.6M NaCl, 0.6M to 0.7M NaCl, 0.7M to 0.8M NaCl, 0.8M to 0.9M NaCl, or 0.9M to 1M NaCl. In some embodiments, the precipitate is washed at least once, at least twice, or at least three times with a buffer. In some embodiments, the precipitate is washed with buffer more than three times. In some embodiments, the fusion protein comprises ranpirnase fused to two or more oligopeptides. In some embodiments, the oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the ranpirnase is a variant ranpirnase. In some embodiments, the purified inclusion bodies are incubated at 95 ℃ after washing with buffer.

In some embodiments, the method of purifying inclusion bodies comprising a fusion protein further comprises washing the precipitate in water. Washing the precipitate with water may remove or reduce any remaining impurities in the solution and any remaining salts or surfactant molecules. In some embodiments, the precipitate is washed at least once, at least twice, or at least three times in water. In some embodiments, the precipitate is washed in water more than three times. In some embodiments, washing the pellet with water results in purified inclusion bodies. In some embodiments, the fusion protein comprises ranpirnase fused to two or more oligopeptides. In some embodiments, the oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the ranpirnase is a variant ranpirnase. In some embodiments, the purified inclusion bodies are incubated at 95 ℃ after washing with water.

In some embodiments, the method of purifying inclusion bodies comprising a fusion protein further comprises solubilizing the inclusion bodies. Solubilization can be performed in the presence or absence of chaotropic agents (e.g., urea or guanidine hydrochloride). In some embodiments, the method of purifying inclusion bodies comprising a fusion protein further comprises solubilizing the inclusion bodies in a solution that does not contain a chaotropic agent. In some embodiments, the method of purifying inclusion bodies comprising a fusion protein further comprises solubilizing the inclusion bodies in a solution comprising a chemical cleavage reagent. In some embodiments, the method of purifying inclusion bodies comprising a fusion protein further comprises solubilizing the inclusion bodies in a solution comprising acid or formic acid. In some embodiments, the inclusion bodies are solubilized in acetic acid. In some embodiments, the inclusion bodies comprise a fusion protein comprising ranpirnase fused to two or more oligopeptides. In some embodiments, the oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the ranpirnase is a variant ranpirnase.

In some embodiments, the method of purifying inclusion bodies comprising a fusion protein comprises purifying the inclusion bodies. Suitable techniques for purification include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, electrophoresis, immunoadsorption, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, liquid Chromatography (LC), high Performance LC (HPLC), fast Performance LC (FPLC), hydroxylapatite chromatography, and lectin chromatography. In some embodiments, the method of purifying inclusion bodies comprising a fusion protein further comprises purifying the inclusion bodies by chromatography. In some embodiments, the method of purifying inclusion bodies comprising the fusion protein does not include purifying the inclusion bodies by chromatography.

In some embodiments, the method of purifying inclusion bodies comprising a fusion protein comprises expressing a fusion protein comprising an oligopeptide operably linked to a protein that forms inclusion bodies in a cell. The protein that forms inclusion bodies in the cell may be any insoluble carrier polypeptide that forms inclusion bodies in the cell. In some embodiments, the protein that forms inclusion bodies in the cell is ranpirnase. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) operably linked to one or more polypeptides via a peptide bond. In some embodiments, the peptide bond is capable of being sequence-specific chemical cleavage. In some embodiments, the oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the fusion protein comprises a cleavable Asp-Pro bond.

In some embodiments, the method of purifying inclusion bodies comprising a fusion protein comprises purifying inclusion bodies comprising a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) operably linked to two or more oligopeptides. In some embodiments, the fusion protein comprises two or more, 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, 15 or more, or 20 or more oligopeptides operably linked to an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide). In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) and one or more oligopeptides operably linked to the N-terminus or C-terminus of the ranpirnase protein. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) and one or more oligopeptides linked to the C-terminus of the ranpirnase. In some embodiments, the one or more oligopeptides linked to the C-terminus of ranpirnase have the same amino acid sequence. In some embodiments, the one or more oligopeptides linked to the C-terminus of ranpirnase have different amino acid sequences. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) and one or more oligopeptides linked to the N-terminus of ranpirnase. In some embodiments, the one or more oligopeptides linked to the N-terminus of ranpirnase have the same amino acid sequence. In some embodiments, the one or more oligopeptides linked to the N-terminus of ranpirnase have different amino acid sequences. In some embodiments, the ranpirnase is a variant ranpirnase.

In some embodiments, the method of purifying inclusion bodies comprising a fusion protein comprises purifying inclusion bodies comprising a fusion polypeptide comprising an oligopeptide operably linked to a protein that forms inclusion bodies in a cell, wherein the operable linkage is a chemically cleavable amino acid sequence. In some embodiments, the two or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising one or more oligopeptides less than 25 amino acids in length. In some embodiments, the one or more oligopeptides are at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 amino acids in length. In some embodiments, one or more oligopeptides are up to 50 amino acids in length. In some embodiments, one or more oligopeptides are less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, or less than 20 amino acids in length. In some embodiments, the one or more oligopeptides are 4 to 50, 6 to 40, 6 to 30, or 8 to 25 amino acids in length. In some embodiments, the fusion polypeptide comprises a cleavable Asp-Pro bond. In some embodiments, the method comprises lysing the cells to form a lysate, centrifuging the cell lysate to form a pellet, and washing the pellet in a surfactant buffer comprising a nonionic surfactant.

In some embodiments, the method of purifying inclusion bodies comprising a fusion protein comprises purifying inclusion bodies comprising a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) operably linked to one or more micropeptides (miPEP). In some embodiments, the one or more oligopeptides comprise miPEP sequences that regulate one or more families of mirnas. In some embodiments, the one or more oligopeptides comprise miPEP sequences that regulate a plant miRNA family. In some embodiments, the method comprises lysing the cells to form a lysate, centrifuging the cell lysate to form a pellet, and washing the pellet in a surfactant buffer comprising a nonionic surfactant.

In some embodiments, the method of purifying inclusion bodies comprising a fusion protein comprises purifying inclusion bodies comprising a fusion polypeptide and further comprising a linker sequence. In some embodiments, the linker sequence is capable of being cleaved by sequence-specific chemical cleavage. In some embodiments, the linker sequence separates one or more oligopeptides and/or the linker sequence separates an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) from one or more oligopeptides. In some embodiments, the linker sequence separating the one or more oligopeptides has the same or different sequence as the linker sequence separating the insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) from the one or more oligopeptides. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a linker sequence comprising the amino acid sequence of SEQ ID NO. 11 that separates one or more oligopeptides from each other and/or separates an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) from one or more oligopeptides. In some embodiments, the inclusion bodies comprise fusion polypeptides comprising a linker sequence comprising an Asp-Pro linker that separates one or more oligopeptides from each other and/or separates an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) from one or more oligopeptides. In some embodiments, the method comprises lysing the cells to form a lysate, centrifuging the cell lysate to form a pellet, and washing the pellet in a surfactant buffer comprising a nonionic surfactant.

In some embodiments, the method of purifying inclusion bodies comprising a fusion protein comprises purifying inclusion bodies comprising a fusion polypeptide comprising a ranpirnase polypeptide comprising the amino acid sequence of SEQ ID NO. 1 operably linked to one or more oligopeptides via peptide bonds. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID No. 1. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the fusion protein comprises a cleavable Asp-Pro bond.

In some embodiments, the method comprises purifying inclusion bodies comprising a fusion polypeptide comprising a variant ranpirnase operably linked to one or more oligopeptides, the variant ranpirnase comprising one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO. 1. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a ranpirnase polypeptide comprising one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO. 1. In some embodiments, the method of purifying inclusion bodies comprising a fusion protein comprises purifying inclusion bodies comprising a fusion polypeptide comprising a ranpirnase polypeptide having one or more amino acid substitutions that reduce the sensitivity of ranpirnase to chemical cleavage as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID NO 1. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a ranpirnase polypeptide having one or more deletions or insertions that reduce the sensitivity of the ranpirnase polypeptide to chemical cleavage as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a ranpirnase polypeptide having one or more amino acid substitutions as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1 that increase yield or promote inclusion body formation. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a ranpirnase polypeptide having one or more deletions or insertions as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1, which one or more deletions or insertions increase yield or promote inclusion body formation. In some embodiments, the method comprises lysing the cells to form a lysate, centrifuging the cell lysate to form a pellet, and washing the pellet in a surfactant buffer comprising a nonionic surfactant.

In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a ranpirnase polypeptide comprising the amino acid sequence of any of SEQ ID NOs 2-10 and 15-22, optionally with 1, 2, 3, 4,5, or all 6C-terminal histidine residues deleted, as shown in fig. 20A. In some embodiments, the inclusion bodies comprise fusion polypeptides comprising a ranpirnase polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs 2-10 and 15-22, optionally with a deletion of 1, 2, 3, 4,5, or all 6C-terminal histidine residues, as shown in fig. 20A. In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide comprising an amino acid sequence that has 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any of SEQ ID NOs 2-10 and 15-22, optionally with a deletion of 1, 2, 3, 4,5, or all 6C-terminal histidine residues, as shown in fig. 20A. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the fusion protein comprises a cleavable Asp-Pro bond. In some embodiments, the method comprises lysing the cells to form a lysate, centrifuging the cell lysate to form a pellet, and washing the pellet in a surfactant buffer comprising a nonionic surfactant.

In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a ranpirnase polypeptide having a modified N-terminus and/or C-terminus operably linked to one or more oligopeptides. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a ranpirnase polypeptide having one or more amino acid substitutions at the N-terminus and/or C-terminus as compared to SEQ ID No. 1. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a ranpirnase polypeptide comprising 11 amino acid substitutions at the N-terminus and/or C-terminus compared to SEQ ID No. 1. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a ranpirnase polypeptide comprising 11 amino acid substitutions at the N-terminus compared to SEQ ID No. 1. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the fusion protein comprises a cleavable Asp-Pro bond. In some embodiments, the method comprises lysing the cells to form a lysate, centrifuging the cell lysate to form a pellet, and washing the pellet in a surfactant buffer comprising a nonionic surfactant.

In some embodiments, the method of purifying inclusion bodies comprising a fusion protein comprises purifying inclusion bodies comprising a fusion polypeptide comprising a TAF12 polypeptide comprising the amino acid sequence of SEQ ID NO. 23 operably linked to one or more oligopeptides via peptide bonds. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a TAF12 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO. 23. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the fusion protein comprises a cleavable Asp-Pro bond.

In some embodiments, the method comprises purifying inclusion bodies comprising a fusion polypeptide comprising a variant TAF12 operably linked to one or more oligopeptides, the variant TAF12 comprising one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO. 23. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a TAF12 polypeptide comprising one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO. 23. In some embodiments, the method of purifying inclusion bodies comprising a fusion protein comprises purifying inclusion bodies comprising a fusion polypeptide comprising a TAF12 polypeptide having one or more amino acid substitutions that reduce the sensitivity of TAF12 to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23. In some embodiments, the inclusion bodies comprise fusion polypeptides comprising a TAF12 polypeptide having one or more deletions or insertions that reduce the sensitivity of the TAF12 polypeptide to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a TAF12 polypeptide having one or more amino acid substitutions as compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23, which one or more amino acid substitutions increases yield or promotes inclusion body formation. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a TAF12 polypeptide having one or more deletions or insertions as compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23, which one or more deletions or insertions increase yield or promote inclusion body formation. In some embodiments, the method comprises lysing the cells to form a lysate, centrifuging the cell lysate to form a pellet, and washing the pellet in a surfactant buffer comprising a nonionic surfactant.

In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a TAF12 polypeptide having a modified N-terminus and/or C-terminus operably linked to one or more oligopeptides. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a TAF12 polypeptide having one or more amino acid substitutions at the N-terminus and/or C-terminus as compared to SEQ ID NO. 23. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus and/or the C-terminus as compared to SEQ ID NO. 23. In some embodiments, the inclusion bodies comprise a fusion polypeptide comprising a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus as compared to SEQ ID NO. 23. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the fusion protein comprises a cleavable Asp-Pro bond. In some embodiments, the method comprises lysing the cells to form a lysate, centrifuging the cell lysate to form a pellet, and washing the pellet in a surfactant buffer comprising a nonionic surfactant.

IV ranpirnase polypeptide

Also provided herein are ranpirnase polypeptides useful as insoluble carrier peptides.

Ranpirnase (onconase), also known as ranpirnase, is an rnase first identified in ranpirus americanus. Ranpirnase is a protein of about 104 amino acids in length, stabilized by four disulfide bonds, and undergoes pH-dependent denaturation. Without being limited by theory, acidic conditions promote denaturation of the ranpirnase, and the denatured form of ranpirnase is highly soluble and efficient renaturation can be achieved only by a method based on reversible blocking of cysteine residues. Under neutral conditions (around pH 7), ranpirnase aggregates readily, and high yields of ranpirnase production can lead to the formation of insoluble aggregates (e.g., inclusion bodies), where the level of protein solubility is so low that it is undetectable. Once the ranpirnase aggregates, the ranpirnase can be removed from the solution by selective precipitation. Furthermore, ranpirnase lacks any aspartate-proline and asparagine-glycine sequences, which makes it resistant to common chemical cleavage strategies.

In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence comprising SEQ ID NO. 1. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 1. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity with SEQ ID No. 1. In some embodiments, the ranpirnase polypeptide is a truncated ranpirnase polypeptide. In some embodiments, the truncation occurs at the N-terminus.

In some embodiments, the ranpirnase polypeptide has one or more amino acid substitutions that reduce its pI. In some embodiments, the ranpirnase polypeptide has a pI of about 8.0, about 7.5, about 7.0, or about 6.5. In some embodiments, the ranpirnase polypeptide has a neutral pI. In some embodiments, the ranpirnase polypeptide has a pI of less than 7.0.

In some embodiments, the ranpirnase polypeptide comprises one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO. 1. In some embodiments, one or more amino acid substitutions is a conservative amino acid substitution. In some embodiments, one or more amino acid substitutions is a non-conservative amino acid substitution. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO. 1. In some embodiments, the ranpirnase polypeptide is an inactivated ranpirnase polypeptide.

In some embodiments, the ranpirnase polypeptide comprises one or more amino acid substitutions that reduce the sensitivity of the ranpirnase polypeptide to chemical cleavage as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, the ranpirnase polypeptide comprises one or more deletions or insertions that reduce the sensitivity of the ranpirnase polypeptide to chemical cleavage as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, the ranpirnase polypeptide has 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid substitutions, insertions, or deletions compared to the ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1.

In some embodiments, the ranpirnase polypeptide comprises one or more amino acid substitutions as compared to the ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1 that increase yield or promote inclusion body formation. In some embodiments, the ranpirnase comprises one or more amino acid substitutions that increase yield or promote inclusion body formation, including substitution of one or more cationic or polar amino acids for non-polar or neutral amino acids. In some embodiments, the one or more amino acid substitutions result in the variant ranpirnase polypeptide having a reduced number of cationic or polar amino acids compared to the ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, the ranpirnase polypeptide comprises one or more deletions or insertions as compared to the ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1, which one or more deletions or insertions increases yield or promotes inclusion body formation. In some embodiments, one or more insertions or deletions that increase yield or promote inclusion body formation result in a variant ranpirnase having a reduced number of cationic or polar amino acids compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1.

In some embodiments, the ranpirnase polypeptide comprises one or more mutations of charged or polar amino acids to nonpolar amino acids. In some embodiments, the ranpirnase polypeptide comprises one or more alanine substitutions. In some embodiments, the ranpirnase polypeptide comprises one or more lysine to alanine substitutions. In some embodiments, the ranpirnase polypeptide comprises 2, 3,4, 5, 6,7, 8, 9, or 10 alanine substitutions. In some embodiments, the ranpirnase polypeptide does not contain lysine residues. In some embodiments, the ranpirnase polypeptide does not contain an arginine residue. In some embodiments, the ranpirnase polypeptide does not contain lysine or arginine residues.

In some embodiments, the ranpirnase polypeptide comprises a modified N-terminus and/or C-terminus. In some embodiments, the ranpirnase polypeptide comprises one or more amino acid substitutions at the N-terminus and/or the C-terminus as compared to SEQ ID No. 1. In some embodiments, the ranpirnase polypeptide comprises 11 amino acid substitutions at the N-terminus and/or the C-terminus as compared to SEQ ID No. 1. In some embodiments, the ranpirnase polypeptide comprises 11 amino acid substitutions at the N-terminus as compared to SEQ ID No. 1.

In some embodiments, the ranpirnase polypeptide comprises the amino acid sequence of any of SEQ ID NOs 2-10, optionally with 1, 2, 3,4, 5, or all 6C-terminal histidine residues deleted, as shown in figure 20A. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence set forth in SEQ ID No. 2. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence set forth in SEQ ID No. 3. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence set forth in SEQ ID No. 4. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence set forth in SEQ ID No. 5. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence set forth in SEQ ID No. 6. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence set forth in SEQ ID No. 7. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence set forth in SEQ ID No. 7. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence set forth in SEQ ID No. 8. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence set forth in SEQ ID No. 9. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence set forth in SEQ ID No. 10. In each case, the above embodiments may optionally have 1,2, 3, 4, 5, or all 6C-terminal histidine residues deleted, as shown in fig. 20A.

In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence tag. In some embodiments, the amino acid sequence tag is a purified amino acid sequence tag. In some embodiments, the amino acid sequence tag is a test amino acid sequence tag. In some embodiments, the tag is less than 20 amino acids in length, less than 10 amino acids in length, or less than 5 amino acids in length. In some embodiments, the tag is 5-20 amino acids in length. In some embodiments, the tag is a hexahistidine or FLAG tag. In some embodiments, the tag is a biotin tag.

In some embodiments, the ranpirnase polypeptide has increased expression levels compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID NO. 1. Expression levels may be measured by any standard technique known in the art. For example, the amount of protein (in grams) can be compared to the amount of total cellular protein (in grams) in a given sample and the measurement determined as a level of recombinant protein per liter. Levels or amounts may also be measured as compared to known standards such as BSA controls. Other techniques for measuring protein levels include, but are not limited to, light absorption analysis of purified protein samples, antibody-based detection (e.g., western blot, FACS, immunofluorescence), activity measurement, and microscopic analysis (e.g., phase contrast, nomads interference, electron microscopy, or fluorescence microscopy). The level or activity can also be compared to a known standard (such as a known amount of purified active protein) for more accurate quantification.

Also provided herein are compositions comprising the ranpirnase mutant polypeptides described herein.

V. fusion polypeptides

Also provided herein are fusion polypeptides comprising a polypeptide that can form inclusion bodies and one or more oligopeptides. Any inclusion body-forming polypeptide disclosed herein can be used. In some embodiments, the inclusion body-forming polypeptide is an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide). In some embodiments, the vector is a variant vector. In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) operably linked to one or more oligopeptides. In some embodiments, one or more oligopeptides are operably linked to an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) by a cleavable bond. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the fusion protein comprises a cleavable Asp-Pro bond.

In some embodiments, the fusion polypeptide comprises two or more, 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, 15 or more, or 20 or more oligopeptides operably linked to an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide). In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) and one or more oligopeptides operably linked to the N-terminus and/or the C-terminus of the ranpirnase protein. In some embodiments, each of the one or more oligopeptides is 4-50 or 5-30 amino acids in length.

In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) and one or more oligopeptides operably linked to the N-terminus or C-terminus of the ranpirnase protein. In some embodiments, the fusion polypeptide comprises 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, 15 or more, or 20 or more oligopeptides operably linked to the N-terminus and/or the C-terminus of an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide). In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) and one or more oligopeptides linked to the C-terminus of ranpirnase. In some embodiments, the one or more oligopeptides linked to the C-terminus of ranpirnase have the same amino acid sequence. In some embodiments, the one or more oligopeptides linked to the C-terminus of ranpirnase have different amino acid sequences. In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) and one or more oligopeptides linked to the N-terminus of ranpirnase. In some embodiments, the one or more oligopeptides linked to the N-terminus of ranpirnase have the same amino acid sequence. In some embodiments, the one or more oligopeptides linked to the N-terminus of ranpirnase have different amino acid sequences.

In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) operably linked to one or more oligopeptides via a cleavable peptide bond. The cleavable bond may be cleaved by an enzyme or chemical reagent. Cleavage of the peptide bond can result in separation of the insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) from the oligopeptide and/or separation of the oligopeptides from each other. In some embodiments, the peptide bond may be cleaved by enzymatic cleavage. In some embodiments, the peptide bond is capable of being sequence-specific chemical cleavage. In some embodiments, the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is with BNPS-skatole, (iii) an aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is with formic acid, (iv) an asparagine-glycine bond and the sequence-specific chemical cleavage is with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is with NTCB. In some embodiments, one or more oligopeptides are operably linked to an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) via a peptide bond comprising an Asp-Pro bond, and the sequence-specific chemical cleavage is performed with formic acid or acetic acid.

In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) operably linked to one or more oligopeptides that are 4-50 or 5-30 amino acids in length. In some embodiments, one or more oligopeptides are less than 25 amino acids in length. In some embodiments, the one or more oligopeptides are at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 amino acids in length. In some embodiments, one or more oligopeptides are up to 50 amino acids in length. In some embodiments, one or more oligopeptides are less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, or less than 20 amino acids in length. In some embodiments, the one or more oligopeptides are 4 to 50, 6 to 40, 6 to 30, or 8 to 25 amino acids in length. In some embodiments, one or more oligopeptides are operably linked to each other through an Asp-Pro linkage. In some embodiments, the operative linkage between the insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) and one or more oligopeptides comprises an Asp-Pro bond. In some embodiments, the insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) is a variant ranpirnase.

In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) operably linked to one or more oligopeptides comprising the amino acid sequence of any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, one or more oligopeptides comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs 12-14 and 31-2401. In some embodiments, one or more oligopeptides comprise an amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the ranpirnase is a variant ranpirnase. In some embodiments, the operable linkage comprises an Asp-Pro bond.

In some embodiments, the fusion polypeptide comprises an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) linked to one or more micro-peptides (miPEP). In some embodiments, the one or more oligopeptides comprise miPEP sequences that regulate the miRNA. In some embodiments, the one or more oligopeptides comprise miPEP sequences that regulate plant, fungal, or metazoan mirnas. In some embodiments, the one or more oligopeptides comprise miPEP sequences that regulate plant mirnas. In some embodiments, the one or more oligopeptides comprise miPEP sequences ：miR156、miR159/319、miR160、miR162、miR164、miR166、miR167、miR168、miR169、miR171、miR172、miR390、miR393、miR394、miR295、miR396、miR397、miR398、miR408、miR403、miR437、miR444 and miR445 family that regulate a plant miRNA selected from the group consisting of. In some embodiments, one or more oligopeptides comprise miPEP sequences having microbial inhibitory activity. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the ranpirnase is a variant ranpirnase. In some embodiments, the operable linkage comprises an Asp-Pro bond.

In some embodiments, the fusion polypeptide further comprises a linker sequence between one or more of the oligopeptides and/or between the insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) and the oligopeptides. The linker sequence may be used as a spacer peptide to separate one or more oligopeptides and/or insoluble carrier polypeptides (e.g., ranpirnase polypeptides or TAF12 polypeptides) from oligopeptides. In some embodiments, the linker sequence provides a cleavable peptide bond. For example, the linker sequence may provide cleavable peptide bonds by incorporating an amino acid sequence that directs sequence-specific cleavage of the fusion polypeptide. In some embodiments, the linker sequence is capable of being cleaved by sequence-specific chemical cleavage. In some embodiments, the fusion polypeptide comprises a linker sequence that separates one or more oligopeptides and/or a linker sequence that separates an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) from one or more oligopeptides. In some embodiments, the linker sequence separating the one or more oligopeptides has the same or different sequence as the linker sequence separating the insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) from the one or more oligopeptides. In some embodiments, the fusion polypeptide includes a linker sequence comprising the amino acid sequence of SEQ ID NO. 11 that separates one or more oligopeptides from each other and/or separates an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) from one or more oligopeptides. In some embodiments, the fusion polypeptide includes a linker sequence comprising an Asp-Pro linker that separates one or more oligopeptides from each other and/or separates an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) from one or more oligopeptides. In some embodiments, sequence-specific chemical cleavage of the linker sequence results in release of the insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) from one or more oligopeptides and/or results in release of one or more oligopeptides from each other.

In some embodiments, the fusion polypeptide further comprises one or more amino acid sequence tags. In some embodiments, the amino acid sequence tag does not affect the solubility of the fusion polypeptide. In some embodiments, the amino acid sequence tag has a neutral pI. In some embodiments, the one or more amino acid sequence tags are purified amino acid sequence tags. Such tag peptides may include, but are not limited to, glutathione-S-transferase (GST), polyhistidine, maltose Binding Protein (MBP), avidin, biotin, streptavidin, histidine (His) tags (e.g., his-6X tags), V5 tags, FLAG tags, influenza Hemagglutinin (HA) tags, myc tags, VSV-G tags, thioredoxin (Trx) tags, and ligands for cellular receptors (e.g., insulin receptor ligands). In some embodiments, one or more amino acid sequence tag sequences are detection or reporting amino acid sequence tags. Examples of reporter amino acid sequence tags include, but are not limited to, horseradish peroxidase (HRP), chloramphenicol Acetyl Transferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, and fluorescent proteins (e.g., GFP, CFP, YFP, BFP, etc.). In some embodiments, one or more amino acid sequence tags are operably linked to the N-terminus and/or the C-terminus of an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) or one or more oligopeptides. In some embodiments, one or more amino acid sequence tags are operably linked to the C-terminus of an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) and the N-terminus of one or more oligopeptides. In some embodiments, one or more amino acid sequence tags are operably linked to the C-terminus of an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) and the N-terminus of one or more oligopeptides. In some embodiments, one or more amino acid sequence tags are operably linked to the C-terminus of one oligopeptide and the N-terminus of another oligopeptide.

In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide comprising the amino acid sequence of SEQ ID NO. 1 operably linked to one or more oligopeptides via peptide bonds. In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 1. In some embodiments, the ranpirnase polypeptide has an amino acid sequence that is 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any of SEQ ID nos. 1. In some embodiments, the ranpirnase polypeptide is a variant ranpirnase polypeptide.

In some embodiments, the fusion polypeptide comprises a variant ranpirnase polypeptide operably linked to one or more oligopeptides through peptide bonds. In some embodiments, the variant ranpirnase comprises one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO. 1. In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO. 1. The one or more insertions or deletions may be of any amino acid length.

In some embodiments, the fusion protein comprises a ranpirnase polypeptide having one or more amino acid substitutions that increase yield or promote inclusion body formation as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, the one or more amino acid substitutions that increase yield or promote inclusion body formation include substitution of one or more cationic or polar amino acids for non-polar or neutral amino acids. In some embodiments, the one or more amino acid substitutions result in the variant ranpirnase polypeptide having a reduced number of cationic or polar amino acids compared to the ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, the fusion protein comprises a ranpirnase polypeptide having one or more deletions or insertions as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1, which one or more deletions or insertions increases yield or promotes inclusion body formation. In some embodiments, one or more insertions or deletions that increase yield or promote inclusion body formation result in a variant ranpirnase having a reduced number of cationic or polar amino acids compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the operable linkage comprises an Asp-Pro bond.

In some embodiments, the fusion protein comprises a ranpirnase polypeptide comprising the amino acid sequence of any of SEQ ID NOs 2-10 and 15-22, optionally with 1,2, 3,4, 5, or all 6C-terminal histidine residues deleted, operably linked to one or more oligopeptides, as shown in FIG. 20A. In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs 2-10 and 15-22, optionally with a deletion of 1,2, 3,4, 5, or all 6C-terminal histidine residues, as shown in fig. 20A. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to SEQ ID NOs 2-10 and 15-22, optionally with a deletion of 1,2, 3,4, 5, or all 6C-terminal histidine residues, as shown in fig. 20A. In some embodiments, the ranpirnase polypeptide comprises one or more amino acid substitutions as compared to SEQ ID NO. 1. In some embodiments, the ranpirnase polypeptide comprises one or more deletions or insertions as compared to SEQ ID NO. 1. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the operable linkage comprises an Asp-Pro bond.

In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide having a modified N-terminus and/or C-terminus operably linked to one or more oligopeptides. In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide having one or more amino acid substitutions at the N-terminus and/or C-terminus as compared to SEQ ID No. 1. In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide comprising 11 amino acid substitutions at the N-terminus and/or C-terminus compared to SEQ ID No. 1. In some embodiments, the fusion polypeptide comprises a ranpirnase polypeptide comprising 11 amino acid substitutions at the N-terminus compared to SEQ ID No. 1. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the operable linkage comprises an Asp-Pro bond.

In some embodiments, the fusion polypeptide comprises a TAF12 polypeptide comprising the amino acid sequence of SEQ ID NO. 23 operably linked to one or more oligopeptides via a peptide bond. In some embodiments, the fusion polypeptide comprises a TAF12 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO. 23. In some embodiments, the TAF12 polypeptide has an amino acid sequence that is 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any one of SEQ ID NOs 23. In some embodiments, the TAF12 polypeptide is a variant TAF12 polypeptide.

In some embodiments, the fusion polypeptide comprises a variant TAF12 polypeptide operably linked to one or more oligopeptides via peptide bonds. In some embodiments, variant TAF12 comprises one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO. 23. In some embodiments, the fusion protein comprises a TAF12 polypeptide comprising one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO. 23. The one or more insertions or deletions may be of any amino acid length.

In some embodiments, the fusion protein comprises a TAF12 polypeptide having one or more amino acid substitutions that increase yield or promote inclusion body formation as compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23. In some embodiments, the one or more amino acid substitutions that increase yield or promote inclusion body formation include substitution of one or more cationic or polar amino acids for non-polar or neutral amino acids. In some embodiments, the one or more amino acid substitutions results in a variant TAF12 polypeptide having a reduced number of cationic or polar amino acids compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23. In some embodiments, the fusion protein comprises a TAF12 polypeptide having one or more deletions or insertions compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23, which one or more deletions or insertions increases yield or promotes inclusion body formation. In some embodiments, one or more insertions or deletions that increase yield or promote inclusion body formation result in a variant TAF12 having a reduced number of cationic or polar amino acids compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the operable linkage comprises an Asp-Pro bond.

In some embodiments, the TAF12 polypeptide comprises one or more amino acid substitutions as compared to SEQ ID NO. 23. In some embodiments, the TAF12 polypeptide comprises one or more deletions or insertions as compared to SEQ ID NO. 23. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the operable linkage comprises an Asp-Pro bond.

In some embodiments, the fusion polypeptide comprises a TAF12 polypeptide having a modified N-terminus and/or C-terminus operably linked to one or more oligopeptides. In some embodiments, the fusion polypeptide comprises a TAF12 polypeptide having one or more amino acid substitutions at the N-terminus and/or C-terminus as compared to SEQ ID NO. 23. In some embodiments, the fusion polypeptide comprises a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus and/or C-terminus as compared to SEQ ID NO. 23. In some embodiments, the fusion polypeptide comprises a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus as compared to SEQ ID NO. 23. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the operable linkage comprises an Asp-Pro bond.

In some embodiments, the fusion polypeptide is expressed in a cell or cell culture. Any known cell or cell culture that produces inclusion bodies is suitable for expressing the fusion peptide. In some embodiments, the fusion polypeptide is expressed in bacteria. In some embodiments, the bacteria are strains of E.coli or Vibrio natrii. In some embodiments, the bacterium is a strain of escherichia coli. In some embodiments, the bacterial cell is a strain lacking Lon and ompT protease functions (e.g., a BL21 escherichia coli strain). In some embodiments, the fusion polypeptide is a yeast cell. In some embodiments, the yeast cell is a strain of pichia pastoris. In some embodiments, the fusion polypeptide is produced by any of the methods disclosed herein.

VI oligopeptides

Provided herein are oligopeptides comprising an active amino acid sequence. In some embodiments, the active amino acid sequence is 4-50 or 5-30 amino acids in length. In some embodiments, the active amino acid sequence is a dipeptide (miPEP). In some embodiments, the oligopeptide comprises the active amino acid sequence and N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid. miPEP is a short peptide (7-44 amino acids long) defined by one or several short open reading frames located in the pri-microRNA sequence of a specific miRNA member. Without being limited by theory, it is believed that miPEP target micrornas (micrornas) encoding them at the transcriptional level, regulating expression of the target pri-micrornas to up-regulate or down-regulate transcription. miPEP can target mirnas involved in various processes, such as mirnas involved in immunity and susceptibility to pathogens (e.g., microorganisms), organogenesis, response to stress, embryonic development, and the like.

In some embodiments, the oligopeptide comprises miPEP sequences that regulate the miRNA. Each miPEP can modulate one or more members in a particular species or modulate a particular miRNA family. miPEP sequences can modulate plant mirnas or metazoan mirnas. In some embodiments, miPEP sequences modulate one or more members of a particular miRNA family. In some embodiments, miPEP sequences modulate plant mirnas. Exemplary plant microrna families that can be regulated by miPEP include, but are not limited to, plant miRNA families miR156、miR159/319、miR160、miR162、miR164、miR166、miR167、miR168、miR169、miR171、miR172、miR390、miR393、miR394、miR295、miR396、miR397、miR398、miR408、miR403、miR437、miR444 and miR445. In some embodiments, the miPEP sequence comprises N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid. In some embodiments, the miPEP sequence has microbial inhibitory activity.

In some embodiments, the oligopeptide comprises an active amino acid sequence having microbial inhibitory activity. In other embodiments, the oligopeptide comprises a peptide microbial inhibitor. In some embodiments, the oligopeptide inhibits the microorganism. In some embodiments, the microorganism is a virus, bacterium, fungus, amoeba, or eukaryote. Exemplary microbial targets that can be inhibited by oligopeptides include, but are not limited to, microorganisms from the following genera: the genera Ceriporia (Venturia), leptosphaeria (Podosphaera), erysiphe (Erysiphe), armillariella (Monolinia), mycosphaerella (Mycosphaerella), leptosphaeria (Uncinula), puccinia (Hemileia), rhizoctonia (Rhizoctonia), puccinia (Puccinia), vitis (Botrytis), helminthosporium (Helminthosporium), rhinococcidiopsis (Rhynchosporium), fusarium (Fusarium), septorius (Septoria), cercospora (Cercospora), alternaria, pyricularia (Pycurospora), pseudomonas (Pseudomonas aeruginosa) Phytophthora (Phytophthora), peronospora (Peronospora), bremia (Bremia), pythum (Pythum), plasmodium (Plasmopara), phytophthora (Scleropthora), plasmodium (Peronosclerospora), rust (Physopella), cercospora (Cercospora), anthrax (Colletotrichum), gibber (Gibberela), torula (Exserohilum), bacillus (Bacillus), trichoderma (Trichoderma), kabatiellu, bipolar (Bipolis), pseudomonas (Pseudomonas Erwinia), mycoplasma (Mycopsma), and Rickettsia (Rickettsia). In some embodiments, the oligopeptide comprises the active amino acid sequence and N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid.

In some embodiments, the oligopeptide is less than 25 amino acids in length. In some embodiments, the oligopeptides are at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 amino acids in length. In some embodiments, the oligopeptides are up to 50 amino acids in length. In some embodiments, the oligopeptide is less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, or less than 20 amino acids in length. In some cases, the oligopeptides are 4 to 50, 6 to 40, 6 to 30, or 8 to 25 amino acids in length. In some embodiments, the oligopeptide comprises the active amino acid sequence and N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid.

In some embodiments, the oligopeptide comprises an active amino acid sequence of 4-50 or 5-30 amino acids in length. In some embodiments, the length of the active amino acid sequence is at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 amino acids. In some embodiments, the oligopeptides are up to 50 amino acids in length. In some embodiments, the active amino acid sequence is less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, or less than 20 amino acids in length. In some cases, the active amino acid sequence is 4 to 50, 6 to 40, 6 to 30, or 8 to 25 amino acids in length. In some embodiments, the oligopeptide comprises the active amino acid sequence and a tag comprising additional amino acids. In some embodiments, the tag is the remainder of the cleavage linker. In some embodiments, the tag is located at the N-terminus or C-terminus of the active amino acid sequence. In some embodiments, the oligopeptide comprises a tag at both the N-terminus and the C-terminus.

In some embodiments, the oligopeptide comprises the amino acid sequence of any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, one or more oligopeptides comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 12. In some embodiments, one or more oligopeptides comprise an amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 13. In some embodiments, one or more oligopeptides comprise at least one amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 14. In some embodiments, the oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the oligopeptide comprises the active amino acid sequence and N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid.

In some embodiments, the oligopeptides provided herein are produced by cleaving concatemers of multiple oligopeptides linked together and expressed as a single polypeptide chain. In some embodiments, the cleavage is cleavage of a cognate concatemer. In some embodiments, the cleavage is cleavage of an heteroconcatemer. In some embodiments, cleavage results in the creation of a short (one to two) amino acid tag at the N-and/or C-terminus of the oligopeptide.

In some embodiments, the oligopeptide comprises the active amino acid sequence and N-terminal and/or C-terminal residues. The N-terminal and/or C-terminal residues may correspond to cleavage tags. For example, the residue may be part of a peptide bond cleaved by chemical cleavage (e.g., acid cleavage) or enzymatic reaction. In some embodiments, the oligopeptide comprises the active amino acid sequence and an N-terminal proline, methionine, tryptophan, glycine or cysteine, and/or a C-terminal methionine, aspartic acid, cysteine, asparagine or tryptophan. In some embodiments, the oligopeptide comprises the active amino acid sequence and N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid. In some embodiments, the oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the active amino acid sequence is miPEP.

VII composition

Provided herein are compositions comprising one or more oligopeptides produced by any of the methods of the present disclosure. In some embodiments, one or more oligopeptides comprise an active amino acid sequence. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the one or more oligopeptides comprise N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid.

In some embodiments, the compositions provided herein are produced by cleavage of a concatemer polypeptide. In some embodiments, the concatemer polypeptide is a heterologous concatemer. In some embodiments, the concatemer polypeptide is a homologous concatemer. In some embodiments, the composition comprises a single type of active oligopeptide generated by cleavage of a cognate concatemer. In some embodiments, the composition comprises a plurality of active oligopeptides produced by cleavage of a heterologous concatemer. Various ratios of peptides in the composition are contemplated, such as 1:1, 1:2, 1:3, 1:4, 1:1:1.

In some embodiments, the composition is formulated as a liquid, gel, emulsion, suspension, capsule, solid, powder, aerosol, paste, coating, spray, soil conditioner (soil drench), microcapsule, emulsifiable concentrate, or granule. In some embodiments, the composition is formulated for agricultural use. In some embodiments, the composition is formulated as a seed treatment, foliar spray, foliar drenching, ready To Use (RTU) formulation, product coating, suspension concentrate, tank mix, aerosol, root dip, soil treatment, dipping formulation, irrigation formulation, or spray formulation. In some embodiments, the composition is formulated to be applied to a plant. In some embodiments, the composition is formulated to be applied to one or more of a seed, root, tuber, fruit, leaf, bulb, rhizome, or flower. In some embodiments, the composition may be applied to the plant by foliar spraying, foliar drenching, drip irrigation, coating, mixing, pouring, dusting, atomizing, soil irrigation, fumigation, soil injection, infiltrating irrigation, spraying, or artificial irrigation. In some embodiments, the composition is formulated to be applied to plants by foliar spraying. In some embodiments, a composition formulated for agricultural use comprises one or more oligopeptides. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the one or more oligopeptides comprise N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid.

In some embodiments, the composition is formulated as a pharmaceutically acceptable composition. In some embodiments, the pharmaceutically acceptable composition is formulated as a liquid, emulsion, liquefied drops, spray, foam formulation, granule, fine particle, powder, capsule, pill, paste, tablet, chew, injection, suppository, cream, shampoo, rinse, resin, aerosol, or bait. In some embodiments, the pharmaceutically acceptable composition is formulated for administration to a non-human animal. In some embodiments, the pharmaceutically acceptable composition is formulated for administration to a human. In some embodiments, the pharmaceutically acceptable composition comprises one or more oligopeptides. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the one or more oligopeptides comprise N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid.

In some embodiments, the composition comprises one or more oligopeptides and a carrier. In some embodiments, the carrier is an agriculturally acceptable carrier. In some embodiments, the agriculturally acceptable carrier includes a solid carrier, a liquid carrier, a gel carrier, a suspension, or an emulsion. In some embodiments, the agriculturally acceptable carrier comprises an adjuvant, inert component, dispersant, surfactant, emulsifier, thickener, wetting agent, fertilizer, mineral, solvent, tackifier, adhesive, or stabilizer. In some embodiments, the composition comprises one or more oligopeptides and a pharmaceutically acceptable carrier. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the one or more oligopeptides comprise N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid.

In some embodiments, the composition comprises one or more oligopeptides comprising an active amino acid sequence. In some embodiments, the active amino acid sequence is a dipeptide (miPEP). In some embodiments, the oligopeptide comprises miPEP sequences that regulate the miRNA. In some embodiments, miPEP sequences modulate one or more members of a particular miRNA family. In some embodiments, miPEP sequences modulate plant mirnas. Exemplary plant microrna families that can be regulated by miPEP include, but are not limited to, plant miRNA families miR156、miR159/319、miR160、miR162、miR164、miR166、miR167、miR168、miR169、miR171、miR172、miR390、miR393、miR394、miR295、miR396、miR397、miR398、miR408、miR403、miR437、miR444 and miR445. In some embodiments, the composition comprises miPEP having microbial inhibitory activity. In some embodiments, the composition comprises miPEP that inhibits a virus, bacterium, fungus, amoeba, or eukaryote. In some embodiments miPEP comprises N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length.

In some embodiments, the composition comprises one or more oligopeptides comprising the amino acid sequence of any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, one or more oligopeptides comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 12. In some embodiments, one or more oligopeptides comprise an amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 13. In some embodiments, one or more oligopeptides comprise at least one amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 14. In some embodiments, the composition comprises two or more oligopeptides. In some embodiments, the composition comprises three or more oligopeptides.

In some embodiments, the composition comprises one or more oligopeptides comprising an active amino acid sequence and N-terminal and/or C-terminal residues. The N-terminal and/or C-terminal residues may correspond to cleavage tags. In some embodiments, the oligopeptide comprises the active amino acid sequence and an N-terminal proline, methionine, tryptophan, glycine or cysteine, and/or a C-terminal methionine, aspartic acid, cysteine, asparagine or tryptophan. In some embodiments, the composition comprises one or more oligopeptides comprising an active amino acid sequence and N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid.

Nucleic acid (VIII)

Nucleic acids encoding any of the fusion polypeptides, insoluble carrier polypeptides (e.g., ranpirnase polypeptides or TAF12 polypeptides) or oligopeptides disclosed herein are also provided. In some embodiments, the nucleic acid comprises a sequence element that enhances expression of the encoded polypeptide in a cell. For example, the nucleic acid may comprise an origin of replication that, when incorporated into a vector, results in a copy number that is high enough to produce expression of the encoded polypeptide. In some embodiments, the nucleic acid comprises a promoter that regulates expression of the fusion polypeptide. In some embodiments, the promoter is a bacterial or yeast promoter. In some embodiments, the promoter is a constitutive or inducible promoter. In some embodiments, the promoter is a strong promoter. In some embodiments, the promoter drives expression of the encoded polypeptide in inclusion bodies.

Any technique known in the art may be used to prepare the nucleic acids of the present disclosure. May include, but is not limited to, cloning, DNA isolation, amplification and purification, enzymatic reactions involving DNA ligases, DNA polymerases, restriction endonucleases, and the like, and various separation techniques such as gel electrophoresis and chromatography. Several standard techniques ：Ausubel et al.(1992) Current Protocols in Molecular Biology, Green/Wiley, New York, N.Y.; Sambrook et al.(1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al.(1982) Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993) Meth.Enzymol.218, Part I; Wu (ed.) (1979) Meth.Enzymol.68; Wu et al.(eds.)(1983) Meth.Enzymol.100 and 101; Grossman and Moldave (eds.)Meth.Enzymol.65; Miller (ed.) (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old and Primrose (1981) Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink (1982) Practical Methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.)(1985) Nucleic Acid Hybridization, IRL Press, Oxford, UK; Setlow and Hollaender (1979) Genetic Engineering：Principles and Methods, Vols. 1-4, Plenum Press, New York; and Ausubel et al (1992) Current Protocols in Molecular Biology, greene/Wiley, new York, N.Y are described below. If used, standard abbreviations and nomenclature in the art are deemed to be used, and abbreviations and nomenclature commonly used in professional journals such as those cited herein.

Also provided herein are vectors comprising nucleic acids encoding any of the fusion polypeptides, insoluble carrier polypeptides (e.g., ranpirnase polypeptides or TAF12 polypeptides) or oligopeptides disclosed herein.

A "vector" is a nucleic acid capable of transporting another nucleic acid. The vector may be, for example, a plasmid, virus, cosmid, or phage. An "expression vector (expression vector)" is a vector that, when present in an appropriate environment, is capable of directing the expression of a protein encoded by one or more genes carried by the vector. Examples of vectors are those that autonomously replicate and express structural gene products in their operably linked DNA segments. Thus, the vector may contain the replicon and selectable markers described above. Vectors include, but are not necessarily limited to, expression vectors. In some embodiments, the vector is a bacterial vector. In some embodiments, the vector is a yeast vector. In some embodiments of the present invention, in some embodiments,

Examples of expression vectors that can be used in prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pET plasmid (Novagen, madison, wis., USA) or pBR322 (ATCC 37017). The pBR322 vector contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells. To construct an expression vector using pBR322, a suitable promoter and DNA sequences encoding one or more polypeptides of the invention are inserted into the pBR322 vector. Other commercially available vectors include, for example, pKK223-3 (PHARMACIA FINE CHEMICALS, uppsala, sweden) and pGEM-1 (Promega Biotec, madison, wis., USA). Other commercially available vectors include those specifically designed for expression of proteins, including pMAL-p2 and pMAL-c2 vectors for expression of proteins fused to maltose binding proteins (NEW ENGLAND Biolabs, beverly, mass., USA).

In yeast, many vectors containing constitutive or inducible promoters can be used. For reviews, see Current Protocols in Molecular Biology, Vol. 2, Ed.Ausubel et al., Greene Publish.Assoc.& Wiley Interscience, Ch. 13 (1988); Bitter et al., Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds.Wu & Grossman, 31987, Acad.Press, N.Y., Vol. 153, pp. 516-544 (1987); Glover, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3 (1986); Bitter, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds.Berger & Kimmel, Acad.Press, N.Y., Vol. 152, pp. 673-684 1987) and The Molecular Biology of the Yeast Saccharomyces, Eds.Strathern et al., Cold Spring Harbor Press, Vols. I and II (1982). may use constitutive yeast promoters such as ADH1 or LEU2 or inducible promoters such as GAL4(Cloning in Yeast, Ch. 3, R. Rothstein In：DNA Cloning Vol. 11, A Practical Approach, Ed.D M Glover, IRL Press, Wash., D.C. (1986)). alternatively vectors may be used that promote integration of the exogenous DNA sequence into the yeast or bacterial chromosome.

Promoter sequences commonly used in recombinant prokaryotic host cell expression vectors include phage T7 promoter (Studier and Moffatt, J. Mol. Biol.189:113 (1986)) \beta-lactamase (penicillinase), lactose promoter system (Chang et al, nature 275:615, 1978; goeddel et al, nature 281:544 (1979)), tryptophan (tap) promoter system (Goeddel et al, nucleic. Acids Res.8:4057 (1980)), EP-A-36776) and tac promoter (manitis, molecular Cloning: A Laboratory Manual, cold Spring Harbor Laboratory; p. 412 (1982)). Particularly useful prokaryotic host cell expression systems employ phage lambda PL promoter and c1857ts thermolabile repressor sequences. Plasmid vectors available from the American Type Culture Collection (ATCC) incorporated into the PL promoter derivatives include plasmid pHUB2 (found in E.coli strain JMB9 (ATCC 37092)) and pPLc28 (found in E.coli RR1 (ATCC 53082)).

In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a protein that forms inclusion bodies in a cell operably linked to one or more oligopeptides via peptide bonds. In some embodiments, the inclusion body tag is a ranpirnase polypeptide. In some embodiments, the protein that forms inclusion bodies in the cell is ranpirnase. In some embodiments, the ranpirnase is a variant ranpirnase.

In some embodiments, the nucleic acid encodes an inclusion body comprising a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) operably linked to one or more polypeptides by peptide bonds. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising two or more, 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, 15 or more, or 20 or more oligopeptides operably linked to an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide). In some embodiments, one or more oligopeptides are operably linked to the N-terminus and/or the C-terminus of ranpirnase. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the fusion polypeptide encoded by the nucleic acid comprises a cleavable Asp-Pro bond.

In some embodiments, the nucleic acid encodes a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) operably linked to one or more polypeptides through peptide bonds capable of being sequence-specific chemical cleavage. In some embodiments, the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is with BNPS-skatole, (iii) an aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is with formic acid, (iv) an asparagine-glycine bond and the sequence-specific chemical cleavage is with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is with NTCB. In some embodiments, one or more oligopeptides are operably linked to an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) via a peptide bond comprising an Asp-Pro bond, and the sequence-specific chemical cleavage is performed with formic acid or acetic acid.

In some embodiments, the nucleic acid encodes a fusion polypeptide comprising one or more oligopeptides less than 25 amino acids in length. In some embodiments, the one or more oligopeptides are at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 amino acids in length. In some embodiments, one or more oligopeptides are up to 50 amino acids in length. In some embodiments, one or more oligopeptides are less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, or less than 20 amino acids in length. In some embodiments, the one or more oligopeptides are 4 to 50, 6 to 40, 6 to 30, or 8 to 25 amino acids in length.

In some embodiments, the nucleic acid encodes a fusion polypeptide comprising ranpirnase operably linked to one or more oligopeptides having the amino acid sequence of any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, one or more oligopeptides comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs 12-14 and 31-2401. In some embodiments, one or more oligopeptides comprise an amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, one or more oligopeptides comprise at least one of the amino acid sequences of any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, one or more oligopeptides comprise at least one amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS.12-14 and 31-2401. In some embodiments, one or more oligopeptides comprise at least one amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, the ranpirnase is a variant ranpirnase. In some embodiments, the one or more oligopeptides comprise an active amino acid sequence and N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid when released from the fusion polypeptide. In some embodiments, the nucleic acid further comprises a linker sequence between one or more of the oligopeptides and/or between the insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) and the oligopeptides. In some embodiments, the linker sequence is capable of being cleaved by sequence-specific chemical cleavage.

In some embodiments, the nucleic acid encodes a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) operably linked to one or more micropeptides (miPEP). In some embodiments, the one or more oligopeptides comprise miPEP sequences that regulate the miRNA. In some embodiments, the one or more oligopeptides comprise miPEP sequences that regulate one or more families of mirnas. In some embodiments, the one or more oligopeptides comprise miPEP sequences that regulate plant mirnas. In some embodiments, one or more oligopeptides comprise miPEP sequences having microbial inhibitory activity.

In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a ranpirnase polypeptide comprising the amino acid sequence of SEQ ID NO. 1 operably linked to one or more oligopeptides via peptide bonds. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 1. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any of SEQ ID nos. 1. In some embodiments, the ranpirnase polypeptide comprises one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO. 1. In some embodiments, the ranpirnase polypeptide comprises one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO. 1.

In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a ranpirnase polypeptide having one or more amino acid substitutions that reduce the sensitivity of the ranpirnase polypeptide to chemical cleavage as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID NO. 1. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a ranpirnase polypeptide having one or more deletions or insertions that reduce the sensitivity of the ranpirnase polypeptide to chemical cleavage as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID NO. 1.

In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a ranpirnase polypeptide having one or more amino acid substitutions, deletions or insertions that increase yield or promote inclusion body formation as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a ranpirnase polypeptide comprising the amino acid sequence of any of SEQ ID NOs 2-10, optionally with 1, 2,3, 4,5, or all 6C-terminal histidine residues deleted, as shown in figure 20A. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs 2-10 and 15-22, optionally with a deletion of 1, 2,3, 4,5, or all 6C-terminal histidine residues, as shown in fig. 20A. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any of SEQ ID NOs 2-10 and 15-22, optionally with a deletion of 1, 2,3, 4,5, or all 6C-terminal histidine residues, as shown in fig. 20A.

In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a ranpirnase polypeptide having a modified N-terminus and/or C-terminus operably linked to one or more oligopeptides. In some embodiments, the ranpirnase polypeptide comprises one or more amino acid substitutions at the N-terminus and/or the C-terminus as compared to SEQ ID No. 1. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a ranpirnase polypeptide comprising 11 amino acid substitutions at the N-terminus and/or C-terminus compared to SEQ ID No. 1. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a ranpirnase polypeptide comprising 11 amino acid substitutions at the N-terminus compared to SEQ ID No. 1. In some embodiments, the nucleic acid further comprises a linker sequence between one or more of the oligopeptides and/or between the ranpirnase polypeptide and the oligopeptides. In some embodiments, the linker sequence is capable of being cleaved by sequence-specific chemical cleavage.

In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a TAF12 polypeptide comprising the amino acid sequence of SEQ ID NO. 23 operably linked to one or more oligopeptides via a peptide bond. In some embodiments, a TAF12 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO. 1. In some embodiments, the TAF12 polypeptide comprises an amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any one of SEQ ID NOs 23. In some embodiments, the TAF12 polypeptide comprises one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO. 23. In some embodiments, the TAF12 polypeptide comprises one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO. 23.

In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a TAF12 polypeptide having one or more amino acid substitutions that reduce the sensitivity of the TAF12 polypeptide to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO. 23. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a TAF12 polypeptide having one or more deletions or insertions that reduce the sensitivity of the TAF12 polypeptide to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO. 23.

In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a TAF12 polypeptide having one or more amino acid substitutions, deletions, or insertions that increase yield or promote inclusion body formation as compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO. 23.

In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a TAF12 polypeptide having a modified N-terminus and/or C-terminus operably linked to one or more oligopeptides. In some embodiments, the TAF12 polypeptide comprises one or more amino acid substitutions at the N-terminus and/or the C-terminus as compared to SEQ ID NO. 23. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus and/or C-terminus as compared to SEQ ID NO. 23. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus as compared to SEQ ID NO. 23. In some embodiments, the nucleic acid further comprises a linker sequence between one or more oligopeptides and/or between the TAF12 polypeptide and the oligopeptide. In some embodiments, the linker sequence is capable of being cleaved by sequence-specific chemical cleavage.

In some embodiments, the nucleic acid encodes an oligopeptide. In some embodiments, the nucleic acid encodes an oligopeptide comprising an active amino acid sequence. In some embodiments, the active amino acid sequence is a dipeptide (miPEP). In some embodiments, the nucleic acid encodes an oligopeptide comprising miPEP sequences that regulate the miRNA. In some embodiments, miPEP sequences modulate one or more members of a particular miRNA family. In some embodiments, miPEP sequences modulate plant mirnas. In some embodiments, miPEP sequences modulate a plant miRNA family ：miR156、miR159/319、miR160、miR162、miR164、miR166、miR167、miR168、miR169、miR171、miR172、miR390、miR393、miR394、miR295、miR396、miR397、miR398、miR408、miR403、miR437、miR444 and miR445 selected from the group consisting of. In some embodiments miPEP comprises N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid.

In some embodiments, the nucleic acid encodes miPEP with microbial inhibitory activity.

In some embodiments, the nucleic acid encodes an oligopeptide comprising the amino acid sequence of any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, the nucleic acid encodes an oligopeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any of SEQ ID NOs 12-14 and 31-2401. In some embodiments, the nucleic acid encodes an oligopeptide comprising an amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, the nucleic acid encodes an oligopeptide comprising at least one of the amino acid sequences of any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, the nucleic acid encodes an oligopeptide comprising at least one amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOS.12-14 and 31-2401. In some embodiments, the nucleic acid encodes an oligopeptide comprising at least one amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any one of SEQ ID NOS 12-14 and 31-2401. In some embodiments, the oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the oligopeptide comprises N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid.

In some embodiments, the nucleic acid encodes a ranpirnase polypeptide. In some embodiments, the ranpirnase is a variant ranpirnase. In some embodiments, the nucleic acid encodes a ranpirnase polypeptide comprising the amino acid sequence of SEQ ID NO. 1. In some embodiments, the nucleic acid encodes a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 1. In some embodiments, the nucleic acid encodes a ranpirnase polypeptide comprising an amino acid sequence that has 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity with SEQ ID No. 1. In some embodiments, the nucleic acid encodes a ranpirnase polypeptide comprising one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO. 1. In some embodiments, the nucleic acid encodes a ranpirnase polypeptide comprising one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO. 1. In some embodiments, the one or more amino acid substitutions or one or more deletions or insertions reduces the sensitivity of the ranpirnase polypeptide to chemical cleavage as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, one or more amino acid substitutions or one or more insertions or deletions result in a variant ranpirnase having increased yield or increased capacity to form inclusion bodies in a cell as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, the nucleic acid encodes a ranpirnase polypeptide comprising the amino acid sequence of any of SEQ ID NOs 2-10 and 15-22, optionally with 1,2, 3, 4, 5, or all 6C-terminal histidine residues deleted, as shown in FIG. 20A. In some embodiments, the nucleic acid encodes a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs 2-10 and 15-22, optionally with a deletion of 1,2, 3, 4, 5, or all 6C-terminal histidine residues, as shown in fig. 20A. In some embodiments, the nucleic acid encodes a ranpirnase polypeptide having at least one amino acid sequence that has 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any one of SEQ ID NOs 12-14 and 31-2401.

In some embodiments, the nucleic acid encodes a ranpirnase polypeptide comprising a modified N-terminus and/or C-terminus. In some embodiments, the nucleic acid encodes a ranpirnase polypeptide comprising one or more amino acid substitutions at the N-terminus and/or C-terminus compared to SEQ ID No. 1. In some embodiments, the nucleic acid encodes a ranpirnase polypeptide comprising 11 amino acid substitutions at the N-terminus and/or C-terminus compared to SEQ ID No. 1. In some embodiments, the nucleic acid encodes a ranpirnase polypeptide comprising 11 amino acid substitutions at the N-terminus compared to SEQ ID No. 1.

In some embodiments, the nucleic acid encodes a TAF12 polypeptide. In some embodiments, TAF12 is a variant TAF12. In some embodiments, the nucleic acid encodes a TAF12 polypeptide comprising the amino acid sequence of SEQ ID NO. 23. In some embodiments, the nucleic acid encodes a TAF12 polypeptide, which TAF12 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO. 23. In some embodiments, the nucleic acid encodes a TAF12 polypeptide, which TAF12 polypeptide comprises an amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to SEQ ID NO. 23. In some embodiments, the nucleic acid encodes a TAF12 polypeptide, which TAF12 polypeptide comprises one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO. 23. In some embodiments, the nucleic acid encodes a TAF12 polypeptide, which TAF12 polypeptide comprises one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO. 23. In some embodiments, the one or more amino acid substitutions or one or more deletions or insertions reduces the sensitivity of the TAF12 polypeptide to chemical cleavage as compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23. In some embodiments, one or more amino acid substitutions or one or more insertions or deletions result in a variant TAF12, which variant TAF12 has an increased yield or an increased ability to form inclusion bodies in a cell compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23. In some embodiments, the nucleic acid encodes a TAF12 polypeptide, which TAF12 polypeptide has at least one amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any one of SEQ ID NOs 12-14 and 31-2401.

In some embodiments, the nucleic acid encodes a TAF12 polypeptide comprising a modified N-terminus and/or C-terminus. In some embodiments, the nucleic acid encodes a TAF12 polypeptide comprising one or more amino acid substitutions at the N-terminus and/or C-terminus as compared to SEQ ID NO. 23. In some embodiments, the nucleic acid encodes a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus and/or C-terminus as compared to SEQ ID NO. 23. In some embodiments, the nucleic acid encodes a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus as compared to SEQ ID NO. 23.

In some embodiments, the nucleic acid encodes an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) that comprises an amino acid sequence tag. In some embodiments, the amino acid sequence tag is a purified amino acid sequence tag. In some embodiments, the amino acid sequence tag is a test amino acid sequence tag. In some embodiments, the ranpirnase is a variant ranpirnase.

IX. cells

Also provided are cells that express any of the fusion polypeptides, oligopeptides, tagged miPEP, or insoluble carrier polypeptides described herein (e.g., ranpirnase polypeptides or TAF12 polypeptides). In some embodiments, the cell is a bacterial or yeast cell. In some embodiments, the cell is a host cell.

In some embodiments, the cell is a bacterial cell, a yeast cell, a plant cell, or a metazoan cell. In some embodiments, the cell is a bacterial cell. In some embodiments, the bacterium is a strain of escherichia coli. In some embodiments, the cell is a cell capable of forming an inclusion body. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is an algal cell.

Microbial host cells useful in the present invention may include, but are not limited to, bacteria such as enterobacteria (e.g., escherichia and salmonella) and Bacillus (Bacillus), acinetobacter (Acinetobacter), streptomyces (Streptomyces), methylobacterium (Methylobacter), rhodococcus (Rhodococcus) and Pseudomonas (Pseudomonas), cyanobacteria (Cyanobacteria) such as Rhodobacter (Rhodobacter) and Rhodococcus (synechinocystis), yeasts (yeasts) such as Saccharomyces (Saccharomyces), zygosaccharomyces (Zygosaccharomyces), kluyveromyces (Kluyveromyces), candida (Candida), hansenula (Hansenula), debaryomyces (Pichia), saccharomyces (yeast), yarrowia (torula) and fungi such as Saccharomyces (Torulopsis) and Aspergillus (Yarrowia) and fungi (e.g., aspergillus and Aspergillus (fungi) such as Saccharomyces).

In some embodiments, the cell comprises one or more modifications to increase expression of the fusion protein. In some embodiments, the cell comprises a mutation or deletion of one or more proteases. In some embodiments, the cell comprises a mutation or deletion in an OmpT or Lon gene.

In some embodiments, the host cell comprises at least one copy of a nucleic acid sequence encoding a fusion polypeptide. At least one copy of the nucleic acid sequence encoding the fusion polypeptide enzyme may be present in the chromosome of a prokaryotic (bacterial) cell or in one chromosome of a eukaryotic cell. Or at least one copy of the nucleic acid sequence encoding the fusion polypeptide may be present in a vector or plasmid present in the cell. As described above, the host cell may be a prokaryotic cell or a eukaryotic cell. If prokaryotic, it may be a bacterial cell. If eukaryotic, it may be a yeast cell, a plant cell or an animal cell. Suitable host cells are described herein.

In some embodiments, the cell expresses a fusion polypeptide comprising an insoluble carrier polypeptide that forms inclusion bodies operably linked to one or more oligopeptides. In some embodiments, the insoluble carrier polypeptide that forms the inclusion body is an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide). In some embodiments, the insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) is a variant insoluble carrier polypeptide (e.g., a variant ranpirnase polypeptide or a variant TAF12 polypeptide). In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, one or more oligopeptides are operably linked to the N-terminus and/or the C-terminus of an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide). In some embodiments, the operable linkage comprises an Asp-Pro bond.

In some embodiments, the cell expresses a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) operably linked to one or more oligopeptides comprising the amino acid sequence of any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, one or more oligopeptides comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs 12-14 and 31-2401. In some embodiments, one or more oligopeptides comprise an amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, one or more oligopeptides comprise at least one of the amino acid sequences of any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, one or more oligopeptides comprise at least one amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOS.12-14 and 31-2401. In some embodiments, one or more oligopeptides comprise at least one amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, the one or more oligopeptides comprise an active amino acid sequence and N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid when released from the fusion polypeptide. In some embodiments, the insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) is a variant insoluble carrier polypeptide (e.g., a variant ranpirnase polypeptide or a variant TAF12 polypeptide). In some embodiments, the operable linkage comprises an Asp-Pro bond.

In some embodiments, the cell expresses a fusion polypeptide comprising an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) operably linked to one or more micropeptides (miPEP). In some embodiments, the one or more oligopeptides comprise miPEP sequences that regulate the miRNA. In some embodiments, the one or more oligopeptides comprise miPEP sequences that regulate one or more members of the miRNA family. In some embodiments, the one or more oligopeptides comprise miPEP sequences that regulate plant mirnas. In some embodiments, the one or more oligopeptides comprise miPEP sequences ：miR156、miR159/319、miR160、miR162、miR164、miR166、miR167、miR168、miR169、miR171、miR172、miR390、miR393、miR394、miR295、miR396、miR397、miR398、miR408、miR403、miR437、miR444 and miR445 family that regulate a plant miRNA selected from the group consisting of. In some embodiments, one or more oligopeptides comprise miPEP sequences having microbial inhibitory activity. In some embodiments, the insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or a TAF12 polypeptide) is a variant insoluble carrier polypeptide (e.g., a variant ranpirnase polypeptide or a variant TAF12 polypeptide). In some embodiments, the operable linkage comprises an Asp-Pro bond.

In some embodiments, the cell expresses a fusion polypeptide further comprising a linker sequence between one or more of the oligopeptides and/or between an insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) and the oligopeptides. In some embodiments, the linker sequence separates one or more oligopeptides and/or the linker sequence separates an insoluble carrier polypeptide (e.g., a ranpirnase polypeptide or TAF12 polypeptide) from one or more oligopeptides. In some embodiments, the linker sequence separating the one or more oligopeptides has the same or different sequence as the linker sequence separating the insoluble carrier polypeptide (e.g., ranpirnase polypeptide or TAF12 polypeptide) from the one or more oligopeptides. In some embodiments, the cell expresses a fusion polypeptide further comprising one or more amino acid sequence tags.

In some embodiments, the cell expresses a fusion polypeptide comprising an insoluble carrier polypeptide comprising a ranpirnase polypeptide. In some embodiments, the ranpirnase polypeptide comprises the amino acid sequence of SEQ ID NO. 1 operably linked to one or more oligopeptides via peptide bonds. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 1. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity with SEQ ID No. 1. In some embodiments, the ranpirnase polypeptide comprises one or more amino acid substitutions, or one or more insertions or deletions, as compared to the amino acid sequence of SEQ ID NO. 1. In some embodiments, the one or more amino acid substitutions or one or more insertions or deletions reduce the sensitivity of the ranpirnase to chemical cleavage as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, one or more amino acid substitutions or one or more insertions or deletions result in a ranpirnase polypeptide having an increased yield or a higher capacity to form inclusion bodies in a cell as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1.

In some embodiments, the ranpirnase polypeptide comprises the amino acid sequence of any of SEQ ID NOs 2-10 and 15-22 operably linked to one or more oligopeptides, optionally with 1,2,3,4, 5, or all 6C-terminal histidine residues deleted, as shown in FIG. 20A. In some embodiments, the cell expresses a fusion polypeptide comprising a ranpirnase polypeptide comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs 2-10 and 15-22, optionally with a deletion of 1,2,3,4, 5, or all 6C-terminal histidine residues, as shown in fig. 20A. In some embodiments, the ranpirnase polypeptide comprises at least one amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any of SEQ ID NOs 1-10 and 15-22, optionally with a deletion of 1,2,3,4, 5, or all 6C-terminal histidine residues, as shown in fig. 20A. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the operable linkage comprises an Asp-Pro bond.

In some embodiments, the ranpirnase polypeptide having a modified N-terminus and/or C-terminus is operably linked to one or more oligopeptides. In some embodiments, the cell expresses a fusion polypeptide comprising a ranpirnase polypeptide having one or more amino acid substitutions at the N-terminus and/or C-terminus as compared to SEQ ID No. 1. In some embodiments, the cell expresses a fusion polypeptide comprising a ranpirnase polypeptide comprising 11 amino acid substitutions at the N-terminus and/or C-terminus compared to SEQ ID No. 1. In some embodiments, the cell expresses a fusion polypeptide comprising a ranpirnase polypeptide comprising 11 amino acid substitutions at the N-terminus compared to SEQ ID No. 1. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the operable linkage comprises an Asp-Pro bond.

In some embodiments, the cell expresses a fusion polypeptide comprising an insoluble carrier polypeptide comprising a TAF12 polypeptide. In some embodiments, the TAF12 polypeptide comprises the amino acid sequence of SEQ ID NO. 23 operably linked to one or more oligopeptides via a peptide bond. In some embodiments, a TAF12 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO. 23. In some embodiments, the TAF12 polypeptide comprises an amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to SEQ ID NO. 23. In some embodiments, the TAF12 polypeptide comprises one or more amino acid substitutions, or one or more insertions or deletions, as compared to the amino acid sequence of SEQ ID NO. 23. In some embodiments, the one or more amino acid substitutions or one or more insertions or deletions reduce the sensitivity of TAF12 to chemical cleavage as compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23. In some embodiments, one or more amino acid substitutions or one or more insertions or deletions result in a TAF12 polypeptide having an increased yield or a higher capacity to form inclusion bodies in a cell compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 23. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the operable linkage comprises an Asp-Pro bond.

In some embodiments, the TAF12 polypeptide has a modified N-terminus and/or C-terminus operably linked to one or more oligopeptides. In some embodiments, the cell expresses a fusion polypeptide comprising a TAF12 polypeptide having one or more amino acid substitutions at the N-terminus and/or C-terminus as compared to SEQ ID NO. 23. In some embodiments, the cell expresses a fusion polypeptide comprising a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus and/or C-terminus as compared to SEQ ID NO. 23. In some embodiments, the cell expresses a fusion polypeptide comprising a TAF12 polypeptide comprising 11 amino acid substitutions at the N-terminus compared to SEQ ID NO. 23. In some embodiments, one or more oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the operable linkage comprises an Asp-Pro bond.

In some embodiments, the cell expresses an oligopeptide. In some embodiments, the oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the cell expresses an oligopeptide comprising the active amino acid sequence. In some embodiments, the active amino acid sequence is miPEP sequences. In some embodiments, miPEP sequences modulate mirnas. In some embodiments, miPEP sequences modulate plant mirnas. In some embodiments miPEP comprises N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid.

In some embodiments, the cell expresses an oligopeptide comprising the amino acid sequence of any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, the oligopeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any of SEQ ID NOs 12-14 and 31-2401. In some embodiments, the oligopeptide comprises an amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, the cell expresses an oligopeptide comprising at least one of the amino acid sequences of any one of SEQ ID NOs 12-14 and 31-2401.

In some embodiments, the oligopeptides are 4-50 or 5-30 amino acids in length. In some embodiments, the oligopeptide comprises the active amino acid sequence and N-terminal proline, C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid.

In some embodiments, the cell expresses a ranpirnase polypeptide. In some embodiments, the ranpirnase polypeptide is a variant ranpirnase polypeptide. In some embodiments, the ranpirnase polypeptide comprises the amino acid sequence of SEQ ID NO. 1. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID No. 1. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity with SEQ ID No. 1. In some embodiments, the cell expresses a ranpirnase polypeptide comprising one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO. 1. In some embodiments, the ranpirnase polypeptide comprises one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO. 1. In some embodiments, the cells express a ranpirnase polypeptide comprising one or more amino acid substitutions that reduce the sensitivity of the ranpirnase polypeptide to chemical cleavage as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 1. In some embodiments, the cells express a ranpirnase polypeptide that comprises one or more deletions or insertions that reduce the sensitivity of the ranpirnase polypeptide to chemical cleavage as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID NO. 1.

In some embodiments, the cells express a ranpirnase polypeptide comprising one or more amino acid substitutions or one or more insertions or deletions that increase yield or promote inclusion body formation as compared to a ranpirnase polypeptide having the amino acid sequence of SEQ ID No. 11.

In some embodiments, the cell expresses a ranpirnase polypeptide comprising the amino acid sequence of any of SEQ ID NOs 2-10 and 15-22, optionally with 1, 2, 3, 4,5, or all 6C-terminal histidine residues deleted, as shown in figure 20A. In some embodiments, the ranpirnase polypeptide comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs 2-10 and 15-22, optionally with a deletion of 1, 2, 3, 4,5, or all 6C-terminal histidine residues, as shown in fig. 20A. In some embodiments, the ranpirnase polypeptide comprises at least one amino acid sequence that has 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, the ranpirnase polypeptide comprises a modified N-terminus and/or C-terminus. In some embodiments, the ranpirnase polypeptide comprises one or more amino acid substitutions at the N-terminus and/or the C-terminus as compared to SEQ ID No. 1. In some embodiments, the ranpirnase polypeptide comprises 11 amino acid substitutions at the N-terminus as compared to SEQ ID No. 1. In some embodiments, the ranpirnase polypeptide comprises a sequence tag.

In some embodiments, the cell expresses a TAF12 polypeptide. In some embodiments, the TAF12 polypeptide is a variant TAF12 polypeptide. In some embodiments, the TAF12 polypeptide comprises the amino acid sequence of SEQ ID NO. 23. In some embodiments, a TAF12 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO. 23. In some embodiments, the TAF12 polypeptide comprises an amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to SEQ ID NO. 23. In some embodiments, the cell expresses a TAF12 polypeptide, which TAF12 polypeptide comprises one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO. 23. In some embodiments, the TAF12 polypeptide comprises one or more insertions or deletions compared to the amino acid sequence of SEQ ID NO. 23. In some embodiments, the cell expresses a TAF12 polypeptide, which TAF12 polypeptide comprises one or more amino acid substitutions that reduce the sensitivity of the TAF12 polypeptide to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO. 23. In some embodiments, the cell expresses a TAF12 polypeptide, which TAF12 polypeptide comprises one or more deletions or insertions that reduce the sensitivity of the TAF12 polypeptide to chemical cleavage compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID NO. 23.

In some embodiments, the cell expresses a TAF12 polypeptide comprising one or more amino acid substitutions or one or more insertions or deletions that increase yield or promote inclusion body formation as compared to a TAF12 polypeptide having the amino acid sequence of SEQ ID No. 11.

In some embodiments, the TAF12 polypeptide comprises at least one amino acid sequence having 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to any one of SEQ ID NOs 12-14 and 31-2401. In some embodiments, the TAF12 polypeptide comprises a modified N-terminus and/or C-terminus. In some embodiments, the TAF12 polypeptide comprises one or more amino acid substitutions at the N-terminus and/or the C-terminus as compared to SEQ ID NO. 23. In some embodiments, the TAF12 polypeptide comprises 11 amino acid substitutions at the N-terminus as compared to SEQ ID NO. 23. In some embodiments, the TAF12 polypeptide comprises a sequence tag.

X-ray kit

Some aspects of the disclosure provide kits comprising any of the fusion polypeptides, insoluble carrier polypeptides (e.g., ranpirnase polypeptides or TAF12 polypeptides) or oligopeptides of the disclosure, any nucleic acid or vector encoding the fusion polypeptides, insoluble carrier polypeptides (e.g., ranpirnase polypeptides or TAF12 polypeptides) or oligopeptides of the disclosure, or any cell expressing the fusion polypeptides, insoluble carrier polypeptides (e.g., ranpirnase polypeptides or TAF12 polypeptides) or oligopeptides of the disclosure.

Kits for use with any of the methods of the present disclosure are also provided herein.

Examples

The following examples further illustrate the invention but should not be construed as in any way limiting its scope. In light of the present disclosure and the level of ordinary skill in the art, it will be appreciated by those skilled in the art that the following embodiments are intended to be exemplary only and that many changes, modifications and alterations may be employed without departing from the scope of the presently disclosed subject matter. The accompanying drawings are intended to be considered as part of the specification and description of the present disclosure.

Example 1 conditions for the Release of peptide from ranpirnase inclusion bodies

This example describes the evaluation of conditions for cleavage of the D-P bond to release peptide from ranpirnase inclusion bodies for production of short peptides in e.

Method of

Release of peptides from ranpirnase

The ranpirnase fusion construct of (FIG. 1A) was expressed in E.coli. Bacterial cells were pelleted, followed by cell pellet lysis by sonication. Cell lysates were pelleted by centrifugation. The pellet was then washed 3 times with Triton X-100 buffer (0.1M Tris, 2% Triton X-100, 2M urea, 10mM EDTA,pH 7.4), 2 times with washing buffer (1M Tris, pH 7.4), and then with water. The purified inclusion bodies were then chemically cleaved by adjustment to the target pH with HCl and incubation at 80 ℃ for 16 hours.

Analysis of peptide expression

Reverse phase high performance liquid chromatography (RP-HPLC) analysis was used to evaluate peptide yield and ranpirnase solubilization (solubilization) (fig. 1B).

Results

In the first experiment, 4 pH's between 2 and 5 were tested (FIGS. 2A-2B). Ranpirnase remains mainly as an inclusion body, but chemical cleavage is still quite efficient. The cleavage efficiency is most efficient in the pH 2 and pH 3 ranges.

A second experiment was performed to determine the optimal balance between efficient pH cleavage and minimal amounts of solubilised fusion protein. Cleavage pH 2.4 to 2.8 is the optimal cleavage condition, with high levels of release peptide and low amounts of solubilised fusion protein (FIGS. 3A-B). Similar results were obtained with sulfuric acid to adjust the pH (data not shown).

To evaluate the effect on amine groups, model peptides containing glutamine residues were tested. As shown in fig. 4A-B, when the cleavage pH is reduced below pH 3.0, the glutamine-containing peptide begins to convert from a basic form to an acidic form, which can be separated by reverse phase HPLC (fig. 4A). This suggests that the conversion of glutamine to glutamic acid under acidic conditions may be achieved by deamidation catalyzed by high temperature and acidic conditions. This observation underscores the importance of working at a pH above 2.6 for asparagine or glutamine bearing pH sensitive peptides.

Example 2 comparison of different inclusion body Forming proteins (inclusion body forming protein)

This example describes the evaluation of different inclusion body forming proteins for the production of short peptides in E.coli.

5 Different inclusion body forming proteins were compared (FIG. 5A), delta (5) -3-ketosteroid isomerase (KSI), the F4 fragment of PurF protein derived from 16.7 kDa (PurF), ompX (OmpX) from E.coli, the histone fold domain (TAF 12) from human transcription factor TAF12 and ranpirnase. All constructs were cloned into pET28a plasmid-based expression systems and transformed into BL21 (DE 3) bacterial strain and selected on kanamycin (50. Mu.g/ml). Expression tests were independently performed 3 times in flasks at 37 ℃ in self-induction medium (fig. 5B). Peptide A peptide yield was estimated by RP-HPLC C18 column using synthetic peptides of the same amino acid sequence at known concentrations tested after chemical cleavage at pH 2.6, 80 ℃ (overnight incubation for 16 hours).

Under the test conditions, all 5 fusion proteins were expressed efficiently and the biologically produced peptides were released.

Example 3 concatemer strategy for peptide production in E.coli (concatemeric strategy).

This example describes the evaluation of concatemer strategies for producing short peptides in E.coli.

Method of

Peptide production using ranpirnase concatamers

Ranpirnase fusion constructs were expressed in E.coli. Bacterial cells were pelleted, followed by cell pellet lysis by sonication. Cell lysates were pelleted by centrifugation. The pellet was then washed 3 times with Triton X-100 buffer (0.1M Tris, 2% Triton X-100, 2M urea, 10 mM EDTA,pH 7.4), 2 times with washing buffer (1M Tris, pH 7.4), and then with water. Purified inclusion bodies were then solubilized by incubation in solubilization buffer (6M guanidine chloride, tris 50mM, 10 mM β -mercaptoethanol, pH 7.4). The solubilised protein was then refolded by extensive dialysis in 0.1M acetic acid at 4 ℃ at pH 3.0 to precipitate ranpirnase, which resulted in guanidine hydrochloride removal. The soluble fraction was cleaved by incubation at 60℃for 24 hours in 0.1M acetic acid at pH 2. After cleavage, ranpirnase was precipitated from the solution by addition of NH ₄ OH buffer (pH 7.0-7.2). The peptide was then lyophilized to remove ammonium acetate, followed by centrifugation.

Peptide expression analysis

Protein expression was assessed by SDS-PAGE analysis of insoluble fractions. Expression testing was performed using BL21 (DE 3) STAR bacterial strain (Invitrogen, reference number C60003) in commercial auto-induction medium (Formedium, reference number AIMTB 02) at 37 ℃.

Results

The concatemer ranpirnase fusion strategy can be used to increase the yield of small peptides (e.g., peptides 10 amino acids long). The concatemer constructs can be designed as homologous concatemers, in which several copies of the same peptide separated by an acid cleavage site are fused to the C-terminus of ranpirnase (fig. 6A), or as heterologous concatemers, in which different peptide sequences are added (fig. 6B).

To determine whether concatemer strategies can be used for efficient peptide production, ranpirnase fusions were prepared with three different 10 amino acid long hydrophilic peptides. Ranpirnase fusions have one or three copies of each peptide. As shown in fig. 7, both the cognate and heterologous concatemer constructs were expressed at high levels. The addition of up to three copies of each peptide did not affect the accumulation of protein in the inclusion bodies in large amounts.

To confirm that the concatemer strategy was applicable to other inclusion body forming proteins, a fusion TAF12 construct with three identical peptides fused to the C-terminus was expressed. The inclusion bodies were washed as in example 3 and then cleaved by adjusting the pH to pH 2.6 with HCl and incubating at 80 ℃. As shown in FIG. 8A, after 4 hours of cleavage, intermediate species corresponding to Shan Tai (mono-peptide), dipeptide (di-peptide) and tripeptide (tri-peptide) species could be detected. With the extension of the cleavage time almost all the material has been converted into the mono-peptide species of interest (fig. 8B).

Example 4 ranpirnase N-terminal modification for increased peptide yield.

This example describes the evaluation of variant ranpirnase proteins for increasing peptide yield and purity.

Method of

Peptide production using ranpirnase concatamers

Peptide production was performed as described in example 3.

Analysis of peptide yield and quality

Reverse phase high performance liquid chromatography (RP-HPLC) analysis was used to evaluate peptide yields. On average three independent purifications were performed on a 50-ml flask scale using self-induction medium.

Peptide mass analysis was performed by RP-HPLC using C18 columns (waters). The main miPEP species were quantified by comparison with injection of known amounts of chemically synthesized PEPTIDE (sb-PEPTIDE) eluting at the same retention time. The yields per liter of culture volume were extrapolated from the average signal detected, and three independent purifications were performed for each construct.

Results

Eight new ranpirnase constructs with variant N-terminal sequences were generated (FIGS. 9A-9B) and the yields of MP18357 peptide (SEQ ID NO: 12) using each construct were compared. Each ranpirnase fusion construct tested had only one copy of MP18357 at the C-terminus (fig. 9A). The mass and quantity of peptides were determined by reverse phase high performance liquid chromatography (RP-HPLC).

Unlike the original ranpirnase sequence, the peptide yield of 3 out of the 8 constructs tested increased by more than 70% (fig. 9C). Constructs a and B (fig. 9B) produced higher amounts of peptide, with similar peptide masses observed with variant ranpirnase compared to the original ranpirnase sequence (fig. 10). Using constructs a and B, the main peptide species eluted at the same elution volume, while no other important species were detected (fig. 10, top and middle panels, interpolation). Construct E represents an unsuccessful N-terminal modification, which resulted in a higher amount of peptide but poor quality (fig. 10, bottom panel). The additional peptide species observed may be due to non-specific cleavage.

Example 5 chemical cleavage to increase the yield and peptide homogeneity of concatemer-generated peptides

Method of

Peptide purification and peptide yield analysis were performed as described in examples 3 and 4, with some modifications. Peptide cleavage was performed by dissolving the soluble fraction in 20% acetic acid at pH 2 and incubating at 40 ℃, 60 ℃ or 80 ℃ for 24 hours. Peptide yields and quality were assessed by RP-HPLC 2, 4 or 20 hours after initiation of incubation.

Results

When acid cleavage was performed in acetic acid at 60 ℃, the use of homologous concatemer ranpirnase fusions carrying three peptide copies resulted in incomplete peptide cleavage (fig. 11A-11B). Additional peaks were detected by RP-HPLC, indicating the presence of uncleaved dipeptide or tripeptide species (FIGS. 11A-11B).

To improve the cleavage efficiency in order to release the cleavage closer to 100%, different incubation times and cleavage temperatures were tested. As shown in fig. 12A, at 40 ℃, incubation time slightly affected cleavage efficiency, and about 25% of peptides were found to be mono-peptides even after 20 hours. It was found that increasing the temperature to 60 ℃ resulted in better cleavage than 40 ℃ resulting in 75% of the peptide being produced as a single peptide species (fig. 12B). Better after cleavage at 80 ℃ for 4 hours than at 60 ℃ and complete cleavage of the peptide was observed after 20 hours (fig. 12C), and only the single peptide fraction was recovered. The quality of the peptide is not affected by the use of the concatemer construct. No further improvement was observed when the acetic acid concentration was varied in the range of 20-50%.

Increasing cleavage temperature improved peptide homogeneity (FIGS. 12A-12C) and peptide yield (FIG. 13) when using concatemer strategy and reduced peptide purification time. The peptide yield at 80 ℃ was about 60% higher than at 60 ℃, mainly due to the higher recovery of the single peptide fraction.

Example 6 direct acetic acid solubilization and development of a chaotropic protocol.

This example describes the development of a chaotropic protocol for peptide production using ranpirnase fusion constructs.

Method of

Peptide purification and peptide yield analysis were performed as described in examples 3 and 4, with some modifications. Direct solubilization of inclusion bodies was performed using protein concentrations of 20 g/L or 50 g/L incubated at 60 ℃ or 80 ℃ in formic acid or acetic acid. Incubation was performed with 20%, 30%, 40% or 50% acetic acid or formic acid (w/v).

Results

Ranpirnase solubility is highly pH dependent and ranpirnase fusion peptide generation strategies are based on this feature. Direct solubilization at acidic pH may be sufficient to solubilize ranpirnase and trigger acidic chemical cleavage directly at 80 ℃.

To test whether acidic pH conditions were sufficient to stabilize ranpirnase and trigger acid cleavage, different concentrations of formic acid or acetic acid were tested for their ability to visually solubilize ranpirnase inclusion bodies and directly effect peptide cleavage. Inclusion bodies (20 g/L at 60 ℃ and 50g/L at 80 ℃) were tested at two concentrations with formic acid or acetic acid ranging from 20-50% (fig. 14A-14B). Even with acetic acid concentrations as low as 20% (w/v) and high protein concentrations of 50g/L, direct solubilization and cleavage of ranpirnase was observed at 80 ℃. When the reaction was performed at 60 ℃, two peptide species were detected, which correspond to the mono-and dipeptide species (fig. 14A). Acetic acid concentration did not affect peptide yield or quality. Increasing the acetic acid concentration appeared to be detrimental because a slight increase in dipeptide content was observed as the acetic acid concentration increased (fig. 14B). FIGS. 14C and 14D show that cleavage with formic acid works as well with acetic acid, but chemical cleavage is less specific than with acetic acid. As shown in fig. 14C, in the case of higher formic acid, formic acid shoulder was observed, exhibiting negative effects. The use of lower acetic acid concentrations further improved direct solubilization and cleavage while maximizing protein concentration.

Example 7 development of a no column downstream purification step.

In this example, the column-free downstream purification step of peptide production using the concatemer strategy was optimized.

Method of

The ranpirnase fusion construct is expressed in bacterial cells. Bacterial cells were pelleted, followed by cell pellet lysis by sonication. Cell lysates were pelleted by centrifugation. The precipitate was then washed 2 times with Triton X-100 buffer at 37℃C (0.05M Tris, 2% Triton X-100, pH 7.5), 2 times with high salt buffer (1M NaCl, pH 12) and heat shock at 95℃for 5 minutes, and finally 2 times with water. Inclusion bodies were then recovered by centrifugation after cooling. The purified inclusion bodies were then solubilized and cleaved in acetic acid. After cleavage, ranpirnase is precipitated from the solution by adding NH ₄ OH to pH 7.0-7.2. The peptide was then lyophilized for removal with ammonium acetate followed by centrifugation.

Results

Many current methods for peptide production require column-based separation methods. However, these methods result in additional steps and increase the cost of peptide production. Thus, the use of a pillarless method to produce peptides presents significant advantages.

To optimize the column-free protocol, an alternative procedure for inclusion body isolation was tested. For example, solubilization with 2% SDS is effective for solubilization of inclusion bodies, but inhibits downstream cleavage and peptide release. Sarkozyl was effective at solubilising inclusion bodies but not under acidic conditions, and thus other conditions were tested.

Washing the inclusion bodies with high concentration of salt (1M NaCl) at alkaline pH followed by heat shock at 95 ℃ to induce DNA unfolding was found to be the most efficient protocol (fig. 15A).

At a pH above the isoelectric point of the protein, the inclusion bodies are negatively charged, which is beneficial to DNA rejection after thermal denaturation. The high salt concentration helps to "shield" the inclusion bodies and avoid attracting nucleic acids after the sample temperature is reduced. Salt alone, heat shock or alkaline pH are not as effective for removing nucleic acids, and a combination of all those parameters is important to reduce nucleic acid contamination during peptide purification.

Example 8 peptide yield was assessed using an optimized peptide production and purification protocol.

This example shows that direct solubilization of inclusion bodies results in higher yields of peptide product.

Method of

Peptide purification using chaotropic agents

The ranpirnase fusion construct is expressed in bacterial cells. Bacterial cells were pelleted, followed by cell pellet lysis by sonication. Cell lysates were pelleted by centrifugation. The pellet was then washed 3 times with Triton X-100 buffer (0.1M Tris, 2% Triton X-100, 2M urea, 10 mM EDTA,pH 7.4), 2 times with washing buffer (1M Tris, pH 7.4), and then with water. Purified inclusion bodies were then solubilized by incubation in solubilization buffer (6M guanidine chloride, tris 50 mM, 10 mM β -mercaptoethanol, pH 7.4). The solubilized protein was then refolded by extensive dialysis at 4 ℃ in 0.1M acetic acid at pH 3.0, which resulted in guanidine hydrochloride removal. The soluble fraction was cleaved by incubation in 0.1M acetic acid at 60 ℃ for 24 hours at pH 2. After cleavage, ranpirnase is precipitated from the solution by adding NH ₄ OH to pH 7.0-7.2. After centrifugation at 10,000 x g for 5 minutes, the aggregated fusion protein was in the pellet and the cleaved peptide was in the supernatant. The peptide was then lyophilized for removal with ammonium acetate.

Chaotropic peptide purification protocol

Ranpirnase fusion is expressed in bacterial cells. Bacterial cells were pelleted, followed by cell pellet lysis by sonication. Cell lysates were pelleted by centrifugation. The precipitate was then washed 2 times with Triton X-100 buffer at 37℃C (0.05M Tris, 2% Triton X-100, pH 7.5), 2 times with high salt buffer (1M NaCl, pH 12) and heat shock at 95℃for 5 minutes, and finally 2 times with water. The purified inclusion bodies were then solubilized and cleaved in acetic acid. After cleavage, ranpirnase is precipitated from the solution by adding NH ₄ OH buffer to pH 7.0-7.2. After centrifugation at 10,000 x g for 5 minutes, the aggregated fusion protein was in the pellet and the cleaved peptide was in the supernatant. The peptide was then lyophilized for removal with ammonium acetate.

Results

To assess whether chaotropic agents-free protocols can produce better yields than protocols requiring the use of chaotropic agents, the yields of peptides produced using each protocol were assessed and compared.

To this end, ranpirnase fusions with one or three (concatemer) peptide copies were used for peptide production according to standard protocols (fig. 15B) or chaotropic protocols (fig. 15A). A chaotropic protocol (fig. 15B), in which the use of chaotropic agents was replaced and inclusion bodies were solubilized directly in 20% acetic acid, resulted in higher yields for all constructs evaluated (fig. 16). Furthermore, in the absence of any column use, increasing the solubilization and cleavage temperatures to 80 ℃ results in high peptide homogeneity and over 80% pure peptide of interest. Because the chaotropic agent-free protocol involves fewer steps, it enables faster and less labor-demanding peptide production, as well as reduced buffer/chemical consumption.

Example 9: for tomato gray mold (Botrytis cinerea) functional assay of (Botrytis cinerea))

In this example, the effect of cleavage scar on peptide function was assessed.

Method of

Peptide production

Peptide production was performed using the chaotropic agent free protocol described in example 8.

Synthetic peptides (MP 18913) mimicking the concatemer sequence with N-terminal proline (P) and C-terminal aspartic acid (D) were chemically synthesized and used as control treatments.

Disease control test

Peptide activity was assessed by disease control assays using tomato plants infected with gray mold (botrytis cinerea). The peptides were sprayed at a concentration of 0.1 g/l or 0.3 g/l 24 hours after infection. The percent disease control was determined based on convex polygon recovery, and a total of 7 plants (n=7) were examined after infection with 250 spores/ml botrytis spores.

Results

Acetic acid cleaves at an aspartic acid-proline (Asp-Pro) peptide bond. Thus, chemical cleavage of ranpirnase concatemer fusions using acetic acid resulted in peptides having N-terminal proline residues and C-terminal aspartic acid residues in addition to the peptide sequence.

To test whether peptides with N-terminal proline and C-terminal aspartic acid amino acids still function, MP18913 (SEQ ID NO: 14) peptides obtained by biological production or chemical synthesis were compared. MP18913 corresponds to a modified MP18357 peptide having an additional proline residue (Pro) at the N-terminus and an aspartic acid residue (Asp) at the C-terminus. Chemically synthesized MP18913 mimics the concatemer sequence by adding proline (P) and aspartic acid (D) at the N-and C-termini of the sequence, respectively. MP18913 was found to be as effective or slightly more effective in controlling botrytis cinerea (Botrytis cinerea) infection than the chemically synthesized version (FIG. 17). Biologically produced MP18913 also provided better protection against tomato gray mold than MP 18357. The bio-produced version of MP18913 (MP 18913-BPD) provides slightly better disease control, with up to 50% disease control, and appears to perform best at 0.3 g/l. The addition of proline and aspartic acid residues did not affect the functionality of the peptide.

Example 10 evaluation of concatemer constructs with N-peptide and C-peptide additions.

This example describes the generation of alternative concatemer constructs for increasing peptide yield.

Method of

Peptide production

Results

As described in example 4, N-terminal modification of ranpirnase fusion is possible and may result in higher peptide yields. Concatemer constructs with peptide copies at the N-terminus and/or additional peptide copies at the C-terminus (fig. 18A) may also lead to increased peptide production.

To test whether ranpirnase concatamers with N-terminal peptides could be used to increase peptide production, constructs were designed that expressed 4 to 6 copies of MP18357 peptides (fig. 18A). These constructs were then expressed in E.coli and evaluated for expression and peptide yield. Using the same expression and purification methods, the expression and peptide yields obtained using these constructs were compared to similar constructs having only peptide copies at the C-terminus.

Results

As shown in fig. 18B and 18C, each ranpirnase fusion protein construct with up to six copies of the oligopeptide was expressed at high levels, whether the peptide was at the N-terminus or the C-terminus. This suggests that even up to six copies of the hydrophilic oligopeptide did not affect ranpirnase carrier activity.

Furthermore, HPLC analysis performed after cleavage showed that chemical cleavage was still efficient with additional copies of oligopeptides at the N and C termini (fig. 19). In fact, for constructs 1x3 and 2x3 comprising the N-terminal oligopeptides, peaks reflecting the individual oligopeptides and smaller peaks representing the individual oligopeptides with methionine were observed.

Example 11 ranpirnase variants with reduced pKa were used to increase peptide yield.

This example describes the evaluation of variant ranpirnase proteins with reduced pKa for increased peptide yield and purity. Without being bound by theory, lowering the pI of the ranpirnase protein can lower the positive charge, thereby reducing the tendency of the fusion protein to bind to negatively charged nucleic acids. Thus, lowering the pI of ranpirnase protein can increase the purity of the fusion protein.

Method of

The ranpirnase fusion construct is expressed in bacterial cells. Bacterial cells were pelleted, followed by cell pellet lysis by sonication. Cell lysates were pelleted by centrifugation. The precipitate was then washed 2 times with Triton X-100 buffer at 37℃C (0.05M Tris, 2% Triton X-100, pH 7.5), 2 times with high salt buffer (1M NaCl, pH 12) and heat shock at 95℃for 5 minutes, and finally 2 times with water. Inclusion bodies were then recovered by centrifugation after cooling. The purified inclusion bodies were then solubilized and cleaved in acetic acid. After cleavage, ranpirnase was precipitated from the solution by addition of NH ₄ OH buffer (pH 7.0-7.2). The peptide was then lyophilized for removal with ammonium acetate followed by centrifugation.

Results

Modification of the ranpirnase sequence to reduce the ranpirnase pKa, for example by mutating lysine residues to alanine residues, will potentially reduce nucleic acid binding and allow washing at lower pH conditions. FIG. 20A shows an alignment of the ranpirnase protein of SEQ ID NO. 1 and variants with three lysine to alanine substitutions. As shown in fig. 20B, the variant with three alanine substitutions had a lower pI (8.78 compared to 9.21). Mutation of three lysine residues in ranpirnase to alanine does not affect ranpirnase expression or its ability to function as a fusion partner.

Additional ranpirnase variants with lower pI were produced, such as those shown in figure 20B. Table 1 shows estimated pI of the ranpirnase of SEQ ID NO. 1 and ranpirnase variants comprising three lysine to alanine substitutions. Table 2 shows the predicted pI of the ranpirnase fusion protein constructs. Fusion proteins comprising oligopeptides fused to ranpirnase variants were expressed and the peptides purified.

Table 1 estimated pI of ranpirnase and ranpirnase variants comprising three lysine to alanine substitutions of SEQ ID NO. 1.

TABLE 2 estimated pI of ranpirnase fusion protein.

The expression levels of the fusion proteins and peptides were compared between ranpirnase variants.

Example 12 materials for scalable bio-fabrication methods.

This example describes raw materials and processes for a cost-effective and highly scalable peptide biological manufacturing process that has been performed according to the methods provided herein.

Results

Large industrial scale peptide production has been achieved using the methods provided herein to perform highly efficient biological processes that take advantage of the physicochemical properties of short, linear and hydrophilic micro-peptides using custom engineered escherichia coli strains that convert simple nutrients to micro-peptides. A well optimized and highly reliable fermentation process ensures high peptide expression after 2-stage batch fermentation.

In the first stage (fig. 21A), involving strain engineering, inclusion bodies produce highly protected peptides via chromosomal integration in e.coli of multiple copies of a nucleic acid sequence encoding a fusion carrier protein linked by a2 amino acid long linker to a concatemer of peptide sequences linked by a2 amino acid long linker. The characteristics of the fusion protein are optimized for high peptide expression, yield and efficient peptide purification. The concatemer strategy was extended to maximize peptide expression by including copies of 2-10 peptide sequences linked by a2 amino acid long linker. Inclusion bodies comprise a very stable matrix of carrier proteins and peptide concatamers. The second stage involves batch growth (fig. 21B).

Sequence listing

Claims

1. A method for producing oligopeptides, comprising:

Expressing a fusion polypeptide, said fusion polypeptide comprising an insoluble carrier polypeptide operably linked to two or more oligopeptides via peptide bonds, and

The two or more oligopeptides are released from the insoluble carrier polypeptide by sequence-specific chemical cleavage of the peptide bonds.

2. The method according to claim 1, wherein the insoluble carrier polypeptide comprises a leopard frog enzyme polypeptide.

3. The method according to claim 1, wherein the insoluble carrier polypeptide comprises the TAF12 polypeptide.

4. The method according to claim 1, wherein the insoluble carrier polypeptide comprises trpΔLE polypeptide, ketosteroid isomerase (KSI) polypeptide, β-galactosidase polypeptide, PagP polypeptide, truncated Escherichia coli PurF F4 fragment polypeptide, Pseudomonas aeruginosa PaP3.30 polypeptide, human transcription factor TAF12 histone folding domain (TAF12-HFD) polypeptide, cleavable self-aggregating tag INTEIN-ELK16, Escherichia coli maltose-binding protein, Escherichia coli RNAse II polypeptide, Escherichia coli alkaline phosphatase polypeptide, Escherichia coli phospholipase A polypeptide, Escherichia coli β-lactamase polypeptide, Salmonella typhimurium MalK protein, Clostridium thermocellulose endoglucanase D polypeptide, Bacillus thuringiensis subsp. aizawai IPL7 insecticidal protein, human cathepsinogen B polypeptide, porcine interferon-γ polypeptide, T5 DNA polymerase polypeptide, and Escherichia coli thioredoxin polypeptide.

5. The method according to any one of claims 1-4, wherein the fusion polypeptide comprises 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, 15 or more oligopeptides, 20 or more oligopeptides.

6. The method according to any one of claims 1-5, wherein the two or more oligopeptides are not identical.

7. The method according to any one of claims 1-6, wherein all oligopeptides are operatively linked to the N-terminus of the insoluble carrier polypeptide, or all oligopeptides are operatively linked to the C-terminus of the insoluble carrier polypeptide.

8. The method according to any one of claims 1-6, wherein at least one oligopeptide is operatively linked to the N-terminus of the insoluble carrier polypeptide, and at least one oligopeptide is operatively linked to the C-terminus of the insoluble carrier polypeptide.

9. The method of claim 8, wherein the fusion polypeptide comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the insoluble carrier polypeptide, and/or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the insoluble carrier polypeptide.

10. The method according to any one of claims 1-9, wherein the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is performed with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is performed with BNPS-skatole, (iii) an aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is performed with formic acid, (iv) an asparagine-glycine bond and the sequence-specific chemical cleavage is performed with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is performed with NTCB.

11. The method according to any one of claims 1-9, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage is performed with acetic acid.

12. The method according to any one of claims 1-11, wherein the oligopeptides are operably linked by peptide bonds and are released from each other when the oligopeptides are released from the insoluble carrier polypeptide.

13. The method according to any one of claims 1-11, wherein the oligopeptides are operably linked by different peptide bonds, and after the oligopeptides are released from the insoluble carrier polypeptide, they are released to each other by sequence-specific chemical cleavage of the different peptide bonds.

14. The method of claim 13, wherein the different peptide bonds comprise (i) methionine and the sequence-specific chemical cleavage is performed with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is performed with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is performed with formic acid, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is performed with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is performed with NTCB.

15. The method of claim 13, wherein the different peptide bonds comprise an Asp-Pro bond and the sequence-specific chemical cleavage is performed with acetic acid.

16. The method according to any one of claims 1-15, wherein the oligopeptide is at least 4 amino acids long, at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long, at least 15 amino acids long, at least 20 amino acids long, or at least 25 amino acids long.

17. The method according to any one of claims 1-16, wherein the oligopeptide is less than 50 amino acids long, less than 45 amino acids long, less than 40 amino acids long, less than 35 amino acids long, less than 30 amino acids long, less than 25 amino acids long, or less than 20 amino acids long.

18. The method according to any one of claims 1-15, wherein the oligopeptide is 4 amino acids to 50 amino acids long, 6 amino acids to 40 amino acids long, 6 amino acids to 30 amino acids long, or 8 amino acids to 25 amino acids long.

19. The method according to any one of claims 1-18, wherein the fusion peptide is expressed in bacteria or yeast.

20. The method of claim 19, wherein the bacteria is Escherichia coli or sodium-dependent Vibrio.

21. The method of claim 19 or claim 20, wherein the yield of the released oligopeptide is at least 10 mg/L bacterial culture, at least 20 mg/L bacterial culture, at least 30 mg/L bacterial culture, at least 40 mg/L bacterial culture, at least 50 mg/L bacterial culture, at least 1 g/L, or at least 5 g/L.

22. A fusion polypeptide comprising an insoluble carrier polypeptide operably linked to two or more oligopeptides, wherein the operable link between the two or more oligopeptides and the insoluble carrier polypeptide comprises a peptide bond capable of sequence-specific chemical cleavage.

23. The fusion polypeptide of claim 22, wherein the insoluble carrier polypeptide comprises a leopard frog enzyme polypeptide.

24. The fusion polypeptide of claim 22, wherein the insoluble carrier polypeptide comprises the TAF12 polypeptide.

25. The fusion polypeptide of claim 22, wherein the insoluble carrier polypeptide comprises trpΔLE polypeptide, ketosteroid isomerase (KSI) polypeptide, β-galactosidase polypeptide, PagP polypeptide, truncated Escherichia coli PurF F4 fragment polypeptide, Pseudomonas aeruginosa PaP3.30 polypeptide, human transcription factor TAF12 histone folding domain (TAF12-HFD) polypeptide, cleavable self-aggregating tag INTEIN-ELK16, Escherichia coli maltose-binding protein, Escherichia coli RNAse II polypeptide, Escherichia coli alkaline phosphatase polypeptide, Escherichia coli phospholipase A polypeptide, Escherichia coli β-lactamase polypeptide, Salmonella typhimurium MalK protein, Clostridium thermocellulose endoglucanase D polypeptide, Bacillus thuringiensis subsp. aizawai IPL7 insecticidal protein, human cathepsinogen B polypeptide, porcine interferon-γ polypeptide, T5 DNA polymerase polypeptide, and Escherichia coli thioredoxin polypeptide.

26. The fusion polypeptide according to any one of claims 22-25, wherein the fusion polypeptide comprises 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, 15 or more oligopeptides, 20 or more oligopeptides.

27. The fusion polypeptide according to any one of claims 22-26, wherein the two or more oligopeptides are not identical.

28. The fusion polypeptide according to any one of claims 22-27, wherein all oligopeptides are operatively linked to the N-terminus of the insoluble carrier polypeptide, or all oligopeptides are operatively linked to the C-terminus of the insoluble carrier polypeptide.

29. The fusion polypeptide according to any one of claims 22-27, wherein at least one oligopeptide is operatively linked to the N-terminus of the insoluble carrier polypeptide, and at least one oligopeptide is operatively linked to the C-terminus of the insoluble carrier polypeptide.

30. The fusion polypeptide of claim 29, wherein the fusion polypeptide comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the insoluble carrier polypeptide, and/or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the insoluble carrier polypeptide.

31. The fusion polypeptide according to any one of claims 22-30, wherein the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is performed with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is performed with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is performed with formic acid, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is performed with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is performed with NTCB.

32. The fusion polypeptide according to any one of claims 22-30, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage is performed with acetic acid.

33. The fusion polypeptide according to any one of claims 22-32, wherein the oligopeptides are operatively linked by the peptide bond and are capable of being released from each other by the sequence-specific chemical cleavage.

34. The fusion polypeptide according to any one of claims 22-32, wherein the oligopeptides are operably linked by different peptide bonds and are capable of being released from each other by different sequence-specific chemical cleavages.

35. The fusion polypeptide of claim 34, wherein the different peptide bonds comprise (i) methionine and the different sequence-specific chemical cleavage is performed with cyanogen bromide, (ii) tryptophan and the different sequence-specific chemical cleavage is performed with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the different sequence-specific chemical cleavage is performed with formic acid, (iv) asparagine-glycine bond and the different sequence-specific chemical cleavage is performed with hydroxylamine, or (v) cysteine and the different sequence-specific chemical cleavage is performed with NTCB.

36. The fusion polypeptide of claim 34, wherein the different peptide bonds comprise an Asp-Pro bond and the different sequence-specific chemical cleavage is performed with acetic acid.

37. The fusion polypeptide according to any one of claims 22-36, wherein the oligopeptide is at least 4 amino acids long, at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long, at least 15 amino acids long, at least 20 amino acids long, or at least 25 amino acids long.

38. The fusion polypeptide according to any one of claims 22-37, wherein the oligopeptide is less than 50 amino acids long, less than 45 amino acids long, less than 40 amino acids long, less than 35 amino acids long, less than 30 amino acids long, less than 25 amino acids long, or less than 20 amino acids long.

39. The fusion polypeptide according to any one of claims 22-36, wherein the oligopeptide is 4 amino acids to 50 amino acids long, 6 amino acids to 40 amino acids long, 6 amino acids to 30 amino acids long, or 8 amino acids to 25 amino acids long.

40. A method for releasing an oligopeptide fused with an insoluble carrier polypeptide that forms inclusion bodies in cells, comprising:

a) Expressing a fusion polypeptide comprising the oligopeptide operably linked to the insoluble carrier polypeptide, wherein the operably linked link is a peptide bond capable of sequence-specific chemical cleavage via acetic acid.

b) Purify the inclusion bodies, and

C) The inclusion bodies are incubated with acid at a temperature greater than 50 °C for at least 1 hour, wherein the oligopeptide is released from the fusion polypeptide by sequence-specific cleavage of the peptide bond.

41. The method of claim 40, wherein the insoluble carrier polypeptide comprises a leopard frog enzyme polypeptide.

42. The method of claim 40, wherein the insoluble carrier polypeptide comprises the TAF12 polypeptide.

43. The method according to claim 40, wherein the insoluble carrier polypeptide comprises trpΔLE polypeptide, ketosteroid isomerase (KSI) polypeptide, β-galactosidase polypeptide, PagP polypeptide, truncated Escherichia coli PurF F4 fragment polypeptide, Pseudomonas aeruginosa PaP3.30 polypeptide, human transcription factor TAF12 histone folding domain (TAF12-HFD) polypeptide, cleavable self-aggregating tag INTEIN-ELK16, Escherichia coli maltose-binding protein, Escherichia coli RNAse II polypeptide, Escherichia coli alkaline phosphatase polypeptide, Escherichia coli phospholipase A polypeptide, Escherichia coli β-lactamase polypeptide, Salmonella typhimurium MalK protein, Clostridium thermocellulose endoglucanase D polypeptide, Bacillus thuringiensis subsp. aizawai IPL7 insecticidal protein, human cathepsinogen B polypeptide, porcine interferon-γ polypeptide, T5 DNA polymerase polypeptide, and Escherichia coli thioredoxin polypeptide.

44. The method according to any one of claims 40-43, wherein the temperature in step c) is greater than 60°C, greater than 70°C, greater than 80°C, or greater than 90°C.

45. The method according to any one of claims 40-44, wherein the temperature in step c) is less than 100 °C, less than 95 °C, less than 90 °C, or less than 85 °C.

46. The method according to any one of claims 40-45, wherein the pH in step c) is less than 3.0.

47. The method according to any one of claims 40-45, wherein the pH in step c) is 2.6 to 2.8.

48. The method according to any one of claims 40-47, wherein the acid is a strong acid, optionally selected from hydrochloric acid and sulfuric acid.

49. The method according to any one of claims 40-47, wherein the acid is a weak acid.

50. The method of claim 49, wherein the weak acid is acetic acid.

51. The method of claim 50, wherein the concentration of acetic acid is at least 2% by weight, at least 3% by weight, at least 4% by weight, or at least 5% by weight.

52. The method according to claim 50 or claim 51, wherein the concentration of acetic acid is less than 50% by weight, less than 45% by weight, less than 40% by weight, less than 35% by weight, or less than 30% by weight.

53. The method according to any one of claims 40-52, wherein the insoluble carrier polypeptide is solubilized by incubation in step c).

54. The method according to any one of claims 40-52, wherein the incubation in step c) does not solubilize the insoluble carrier polypeptide.

55. The method according to any one of claims 40-54, wherein the fusion polypeptide comprises 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, 15 or more oligopeptides, 20 or more oligopeptides.

56. The method according to any one of claims 40-55, wherein the two or more oligopeptides are not identical.

57. The method according to any one of claims 40-56, wherein all oligopeptides are operatively linked to the N-terminus of the insoluble carrier polypeptide, or all oligopeptides are operatively linked to the C-terminus of the insoluble carrier polypeptide.

58. The method according to any one of claims 40-56, wherein at least one oligopeptide is operatively linked to the N-terminus of the insoluble carrier polypeptide, and at least one oligopeptide is operatively linked to the C-terminus of the insoluble carrier polypeptide.

59. The method of claim 58, wherein the fusion polypeptide comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, and/or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, operably linked to the C-terminus of the insoluble carrier polypeptide.

60. The method according to any one of claims 40-59, wherein the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is performed with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is performed with BNPS-skatole, (iii) an aspartic-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is performed with formic acid, (iv) an asparagine-glycine bond and the sequence-specific chemical cleavage is performed with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is performed with NTCB.

61. The method according to any one of claims 40-60, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage is an acid cleavage.

62. The method according to any one of claims 40-61, wherein the oligopeptides are operably linked by peptide bonds and are released from each other when the oligopeptides are released from the insoluble carrier polypeptide.

63. The method according to any one of claims 40-62, wherein the oligopeptides are operably linked by different peptide bonds, and after the oligopeptides are released from the insoluble carrier polypeptide, they are released from each other by sequence-specific chemical cleavage of the different peptide bonds.

64. The method of claim 63, wherein the different peptide bonds comprise (i) methionine and the sequence-specific chemical cleavage is performed with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is performed with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is performed with formic acid, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is performed with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is performed with NTCB.

65. The method of claim 64, wherein the different peptide bonds comprise an Asp-Pro bond and the sequence-specific chemical cleavage is an acid cleavage.

66. The method according to any one of claims 40-65, wherein the oligopeptide is at least 4 amino acids long, at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long, at least 15 amino acids long, at least 20 amino acids long, or at least 25 amino acids long.

67. The method according to any one of claims 40-66, wherein the oligopeptide is less than 50 amino acids long, less than 45 amino acids long, less than 40 amino acids long, less than 35 amino acids long, less than 30 amino acids long, less than 25 amino acids long, or less than 20 amino acids long.

68. The method according to any one of claims 40-65, wherein the oligopeptide is 4 amino acids to 50 amino acids long, 6 amino acids to 40 amino acids long, 6 amino acids to 30 amino acids long, or 8 amino acids to 25 amino acids long.

69. The method according to any one of claims 40-68, wherein the fusion peptide is expressed in bacteria or yeast.

70. The method of claim 69, wherein the bacteria is Escherichia coli or sodium-dependent Vibrio.

71. A method for purifying inclusion bodies containing a fusion protein, the method comprising:

a) Expressing a fusion protein comprising an oligopeptide operatively linked to an insoluble carrier polypeptide that forms inclusion bodies in cells, wherein the operative link is a chemically cleavable amino acid sequence.

b) Lyse the cells to form cell lysates.

c) Centrifuge the cell lysate to form a precipitate.

d) Wash the precipitate at least once, at least twice, or at least three times in a surfactant buffer containing a nonionic surfactant.

e) Wash the precipitate at least once, at least twice, or at least three times in a salt buffer containing at least 0.5 M NaCl, and

f) Wash the precipitate in water at least once, at least twice, or at least three times to produce purified inclusion bodies.

72. The method of claim 71, wherein the insoluble carrier polypeptide comprises the TAF12 polypeptide.

73. The method of claim 71, wherein the insoluble carrier polypeptide comprises a leopard frog enzyme polypeptide.

74. The method according to claim 71, wherein the insoluble carrier polypeptide comprises trpΔLE polypeptide, ketosteroid isomerase (KSI) polypeptide, β-galactosidase polypeptide, PagP polypeptide, truncated Escherichia coli PurF F4 fragment polypeptide, Pseudomonas aeruginosa PaP3.30 polypeptide, human transcription factor TAF12 histone folding domain (TAF12-HFD) polypeptide, cleavable self-aggregating tag INTEIN-ELK16, Escherichia coli maltose-binding protein, Escherichia coli RNase II polypeptide, Escherichia coli alkaline phosphatase polypeptide, Escherichia coli phospholipase A polypeptide, Escherichia coli β-lactamase polypeptide, Salmonella typhimurium MalK protein, Clostridium thermocellulose endoglucanase D polypeptide, Bacillus thuringiensis subsp. aizawai IPL7 insecticidal protein, human cathepsinogen B polypeptide, porcine interferon-γ polypeptide, T5 DNA polymerase polypeptide, and Escherichia coli thioredoxin polypeptide.

75. The method according to any one of claims 71-74, wherein the salt buffer is at least 0.6 M NaCl, at least 0.7 M NaCl, or at least 0.75 M NaCl.

76. The method according to any one of claims 71-75, wherein the fusion polypeptide comprises 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, 15 or more oligopeptides, 20 or more oligopeptides.

77. The method according to any one of claims 71-76, wherein the two or more oligopeptides are not identical.

78. The method according to any one of claims 71-77, wherein all oligopeptides are operatively linked to the N-terminus of the insoluble carrier polypeptide, or all oligopeptides are operatively linked to the C-terminus of the insoluble carrier polypeptide.

79. The method according to any one of claims 71-77, wherein at least one oligopeptide is operatively linked to the N-terminus of the insoluble carrier polypeptide, and at least one oligopeptide is operatively linked to the C-terminus of the insoluble carrier polypeptide.

80. The method of claim 79, wherein the fusion polypeptide comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, and/or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides, operably linked to the C-terminus of the insoluble carrier polypeptide.

81. The method according to any one of claims 71-80, wherein the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is performed with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is performed with BNPS-skatole, (iii) an aspartic-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is performed with formic acid, (iv) an asparagine-glycine bond and the sequence-specific chemical cleavage is performed with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is performed with NTCB.

82. The method according to any one of claims 71-80, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage is an acid cleavage.

83. The method according to any one of claims 71-82, wherein the oligopeptides are operably linked by peptide bonds and are released from each other when the oligopeptides are released from the insoluble carrier polypeptide.

84. The method according to any one of claims 71-82, wherein the oligopeptides are operably linked by different peptide bonds, and after the oligopeptides are released from the insoluble carrier polypeptide, they are released to each other by sequence-specific chemical cleavage of the different peptide bonds.

85. The method of claim 84, wherein the different peptide bonds comprise (i) methionine and the sequence-specific chemical cleavage is performed with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is performed with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is performed with formic acid, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is performed with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is performed with NTCB.

86. The method of claim 84, wherein the different peptide bonds comprise an Asp-Pro bond and the sequence-specific chemical cleavage is performed with acetic acid.

87. The method according to any one of claims 71-86, wherein the oligopeptide is at least 4 amino acids long, at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long, at least 15 amino acids long, at least 20 amino acids long, or at least 25 amino acids long.

88. The method according to any one of claims 71-87, wherein the oligopeptide is less than 50 amino acids long, less than 45 amino acids long, less than 40 amino acids long, less than 35 amino acids long, less than 30 amino acids long, less than 25 amino acids long, or less than 20 amino acids long.

89. The method according to any one of claims 71-86, wherein the oligopeptide is 4 amino acids to 50 amino acids long, 6 amino acids to 40 amino acids long, 6 amino acids to 30 amino acids long, or 8 amino acids to 25 amino acids long.

90. The method according to any one of claims 71-89, wherein the fusion peptide is expressed in bacteria or yeast.

91. The method of claim 90, wherein the bacteria is Escherichia coli or sodium-dependent Vibrio.

92. A method for producing a fusion polypeptide, comprising:

The expression of a fusion polypeptide comprising an oligopeptide operably linked to the C-terminus of a leopard enzyme polypeptide, wherein the leopard enzyme polypeptide contains one or more amino acid substitutions in the 11 N-terminal amino acids compared to SEQ ID NO: 1, wherein when expressed under the same conditions, the leopard enzyme-oligopeptide fusion protein comprising the leopard enzyme polypeptide is expressed at a higher level than the leopard enzyme fusion protein comprising SEQ ID NO: 1.

93. The method of claim 92, wherein the fusion polypeptide comprises 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, 15 or more oligopeptides, 20 or more oligopeptides.

94. The method according to claim 92 or claim 95, wherein the two or more oligopeptides are not identical.

95. The fusion polypeptide according to any one of claims 92-94, wherein all oligopeptides are operatively linked to the N-terminus of the leopard frog enzyme polypeptide, or all oligopeptides are operatively linked to the C-terminus of the leopard frog enzyme polypeptide.

96. The method according to any one of claims 92-94, wherein at least one oligopeptide is operatively linked to the N-terminus of the leopard frog enzyme polypeptide, and at least one oligopeptide is operatively linked to the C-terminus of the leopard frog enzyme polypeptide.

97. The method of claim 96, wherein the fusion polypeptide comprises 2 or more oligopeptides, 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides operably linked to the N-terminus of the leopard enzyme polypeptide, and/or 2 or more oligopeptides, 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides operably linked to the C-terminus of the leopard enzyme polypeptide.

98. The method according to any one of claims 92-97, wherein the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is performed with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is performed with BNPS-skatole, (iii) an aspartic-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is performed with formic acid, (iv) an asparagine-glycine bond and the sequence-specific chemical cleavage is performed with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is performed with NTCB.

99. The method according to any one of claims 92-97, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage is performed with acetic acid.

100. The method according to any one of claims 92-99, wherein the oligopeptides are operably linked by peptide bonds and are released from each other when the oligopeptides are released from the leopard frog enzyme.

101. The method according to any one of claims 92-99, wherein the oligopeptides are operably linked by different peptide bonds, and after the oligopeptides are released from the leopard frog enzyme, they are released from each other by sequence-specific chemical cleavage of the different peptide bonds.

102. The method of claim 101, wherein the different peptide bonds comprise (i) methionine and the sequence-specific chemical cleavage is performed with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is performed with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is performed with formic acid, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is performed with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is performed with NTCB.

103. The method of claim 101, wherein the different peptide bonds comprise an Asp-Pro bond and the sequence-specific chemical cleavage is an acid cleavage.

104. The method according to any one of claims 92-103, wherein the oligopeptide is at least 4 amino acids long, at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long, at least 15 amino acids long, at least 20 amino acids long, or at least 25 amino acids long.

105. The method according to any one of claims 92-104, wherein the oligopeptide is less than 50 amino acids long, less than 45 amino acids long, less than 40 amino acids long, less than 35 amino acids long, less than 30 amino acids long, less than 25 amino acids long, or less than 20 amino acids long.

106. The method according to any one of claims 92-103, wherein the oligopeptide is 4 amino acids to 50 amino acids long, 6 amino acids to 40 amino acids long, 6 amino acids to 30 amino acids long, or 8 amino acids to 25 amino acids long.

107. The method according to any one of claims 92-106, wherein the fusion peptide is expressed in bacteria or yeast.

108. The method of claim 107, wherein the bacteria is Escherichia coli or sodium-dependent Vibrio.

109. A leopard frog enzyme polypeptide, wherein, compared with SEQ ID NO: 1, the leopard frog enzyme polypeptide contains one or more amino acid substitutions in the 11 N-terminal amino acids of the leopard frog enzyme polypeptide, wherein, when expressed under the same conditions, the leopard frog enzyme polypeptide is expressed at a higher level than the leopard frog enzyme protein of SEQ ID NO: 1.

110. The leopard frog enzyme polypeptide according to claim 109, wherein the leopard frog enzyme polypeptide comprises an amino acid sequence of one of SEQ ID NO: 2-10 and 15-22.

111. A fusion polypeptide comprising a leopard enzyme polypeptide operably linked to one or more oligopeptides, wherein the operable link between the one or more oligopeptides and the leopard enzyme polypeptide comprises a peptide bond capable of sequence-specific chemical cleavage, wherein the leopard enzyme polypeptide contains one or more amino acid substitutions in the 11 N-terminal amino acids of the leopard enzyme polypeptide compared to SEQ ID NO: 1, wherein the leopard enzyme polypeptide is expressed at a higher level than the leopard enzyme protein of SEQ ID NO: 1 when expressed under the same conditions.

112. The fusion polypeptide according to claim 111, wherein the leopard frog enzyme polypeptide comprises an amino acid sequence of one of SEQ ID NO: 2-10 and 15-22.

113. The fusion polypeptide of claim 111 or claim 112, wherein the fusion polypeptide comprises 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, 15 or more oligopeptides, 20 or more oligopeptides.

114. The fusion polypeptide according to any one of claims 111-113, wherein the two or more oligopeptides are not identical.

115. The fusion polypeptide according to any one of claims 111-114, wherein all oligopeptides are operatively linked to the N-terminus of the leopard frog enzyme polypeptide, or all oligopeptides are operatively linked to the C-terminus of the leopard frog enzyme polypeptide.

116. The fusion polypeptide according to any one of claims 111-114, wherein at least one oligopeptide is operatively linked to the N-terminus of the leopard frog enzyme polypeptide, and at least one oligopeptide is operatively linked to the C-terminus of the leopard frog enzyme polypeptide.

117. The fusion polypeptide of claim 116, wherein the fusion polypeptide comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more oligopeptides operably linked to the N-terminus of the leopard enzyme polypeptide, and/or 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more oligopeptides operably linked to the C-terminus of the leopard enzyme polypeptide.

118. The fusion polypeptide according to any one of claims 111-117, wherein the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is performed with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is performed with BNPS-skatole, (iii) an aspartic-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is performed with formic acid, (iv) an asparagine-glycine bond and the sequence-specific chemical cleavage is performed with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is performed with NTCB.

119. The fusion polypeptide according to any one of claims 111-117, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage is an acid cleavage.

120. The fusion polypeptide according to any one of claims 111-119, wherein the oligopeptides are operatively linked by the peptide bond and are capable of being released from each other by the sequence-specific chemical cleavage.

121. The fusion polypeptide according to any one of claims 105-119, wherein the oligopeptides are operably linked by different peptide bonds and are capable of being released from each other by different sequence-specific chemical cleavages.

122. The fusion polypeptide of claim 121, wherein the different peptide bonds comprise (i) methionine and the different sequence-specific chemical cleavage is performed with cyanogen bromide, (ii) tryptophan and the different sequence-specific chemical cleavage is performed with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the different sequence-specific chemical cleavage is performed with formic acid, (iv) asparagine-glycine bond and the different sequence-specific chemical cleavage is performed with hydroxylamine, or (v) cysteine and the different sequence-specific chemical cleavage is performed with NTCB.

123. The fusion polypeptide of claim 121, wherein the different peptide bonds comprise an Asp-Pro bond and the different sequence-specific chemical cleavages are acid cleavages.

124. The fusion polypeptide according to any one of claims 111-123, wherein the oligopeptide is at least 4 amino acids long, at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long, at least 15 amino acids long, at least 20 amino acids long, or at least 25 amino acids long.

125. The fusion polypeptide according to any one of claims 111-124, wherein the oligopeptide is less than 50 amino acids long, less than 45 amino acids long, less than 40 amino acids long, less than 35 amino acids long, less than 30 amino acids long, less than 25 amino acids long, or less than 20 amino acids long.

126. The fusion polypeptide according to any one of claims 111-125, wherein the oligopeptide is 4 amino acids to 50 amino acids long, 6 amino acids to 40 amino acids long, 6 amino acids to 30 amino acids long, or 8 amino acids to 25 amino acids long.

127. An oligopeptide comprising an active amino acid sequence and an N-terminal proline, a C-terminal aspartic acid, or both N-terminal proline and C-terminal aspartic acid, wherein the active amino acid sequence is a miPEP and the oligopeptide regulates miRNA, or the active amino acid sequence is a peptide microbial inhibitor and the oligopeptide inhibits microorganisms.

128. A nucleic acid encoding a fusion polypeptide according to any one of claims 22-39, a leopard frog enzyme according to claim 109 or claim 110, or a fusion polypeptide according to any one of claims 111-126.

129. A cell comprising the nucleic acid according to claim 128.

130. The cell of claim 129, wherein the cell is a bacterial cell or a yeast cell.

131. The cell of claim 129, wherein the bacteria is Escherichia coli or sodium-dependent Vibrio.

132. The cell of claim 131, wherein the cell is a BL21 bacterial cell.

133. The cell of claim 131, wherein the cell does not express Lon and ompT proteases.

134. The nucleic acid according to claim 128, wherein the nucleic acid is an isolated nucleic acid.

135. The method according to any one of claims 1-108, wherein the fusion polypeptide or fusion peptide is expressed in Escherichia coli.

136. The method according to any one of claims 1-108 and 135, wherein the fusion polypeptide or fusion protein is expressed in cells grown in a fermentation bioreactor.

137. The method according to any one of claims 1-108, wherein the cutting is performed at a pH of about 2-3.5 and a temperature of about 70-90 °C for about 1-24 hours.

138. A method for producing a fusion polypeptide, comprising:

Expressing a fusion polypeptide comprising an oligopeptide operatively linked to the C-terminus or C-terminus of a modified TAF polypeptide, wherein the modified TAF polypeptide comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 23.

139. The method of claim 138, wherein the fusion polypeptide comprises 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, 15 or more oligopeptides, 20 or more oligopeptides.

140. The method of claim 138 or claim 139, wherein the two or more oligopeptides are not identical.

141. The method according to any one of claims 138-140, wherein all oligopeptides are operatively linked to the N-terminus of the modified TAF polypeptide, or all oligopeptides are operatively linked to the C-terminus of the modified TAF polypeptide.

142. The method according to any one of claims 138-140, wherein at least one oligopeptide is operatively linked to the N-terminus of the modified TAF polypeptide, and at least one oligopeptide is operatively linked to the C-terminus of the modified TAF polypeptide.

143. The method of claim 142, wherein the fusion polypeptide comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the modified TAF polypeptide, and/or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the modified TAF polypeptide.

144. The method according to any one of claims 138-143, wherein the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is performed with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is performed with BNPS-skatole, (iii) an aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is performed with formic acid, (iv) an asparagine-glycine bond and the sequence-specific chemical cleavage is performed with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is performed with NTCB.

145. The method according to any one of claims 138-143, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage is performed with acetic acid or sulfuric acid.

146. The method according to any one of claims 138-145, wherein the oligopeptides are operably linked by peptide bonds and are released from each other when the oligopeptides are released from the modified TAF polypeptide.

147. The method according to any one of claims 138-145, wherein the oligopeptides are operatively linked by different peptide bonds, and after the oligopeptides are released from the modified TAF polypeptide, they are released from each other by sequence-specific chemical cleavage of the different peptide bonds.

148. The method of claim 147, wherein the different peptide bonds comprise (i) methionine and the sequence-specific chemical cleavage is performed with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is performed with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is performed with formic acid, (iv) asparagine-glycine bond and the sequence-specific chemical cleavage is performed with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is performed with NTCB.

149. The method of claim 147, wherein the different peptide bonds comprise an Asp-Pro bond and the sequence-specific chemical cleavage is an acid cleavage.

150. The method according to any one of claims 138-149, wherein the oligopeptide is at least 4 amino acids long, at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long, at least 15 amino acids long, at least 20 amino acids long, or at least 25 amino acids long.

151. The method according to any one of claims 138-150, wherein the oligopeptide is less than 50 amino acids long, less than 45 amino acids long, less than 40 amino acids long, less than 35 amino acids long, less than 30 amino acids long, less than 25 amino acids long, or less than 20 amino acids long.

152. The method according to any one of claims 138-149, wherein the oligopeptide is 4 amino acids to 50 amino acids long, 6 amino acids to 40 amino acids long, 6 amino acids to 30 amino acids long, or 8 amino acids to 25 amino acids long.

153. The method according to any one of claims 138-152, wherein the fusion peptide is expressed in bacteria or yeast.

154. The method of claim 153, wherein the bacteria is Escherichia coli or sodium-dependent Vibrio.

155. A modified TAF polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 23.

156. The modified TAF polypeptide according to claim 155, wherein the modified TAF polypeptide comprises the amino acid sequence of SEQ ID NO: 23.

157. A fusion polypeptide comprising a modified TAF polypeptide operably linked to one or more oligopeptides, wherein the operable link between the one or more oligopeptides and the modified TAF polypeptide comprises a peptide bond capable of sequence-specific chemical cleavage, wherein the modified TAF polypeptide comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 23.

158. The fusion polypeptide of claim 157, wherein the modified TAF polypeptide comprises the amino acid sequence of SEQ ID NO: 23.

159. The fusion polypeptide of claim 157 or claim 158, wherein the fusion polypeptide comprises 3 or more oligopeptides, 4 or more oligopeptides, 5 or more oligopeptides, 6 or more oligopeptides, 8 or more oligopeptides, 10 or more oligopeptides, 15 or more oligopeptides, 20 or more oligopeptides.

160. The fusion polypeptide according to any one of claims 157-159, wherein the two or more oligopeptides are not identical.

161. The fusion polypeptide according to any one of claims 157-160, wherein all oligopeptides are operatively linked to the N-terminus of the modified TAF polypeptide, or all oligopeptides are operatively linked to the C-terminus of the modified TAF polypeptide.

162. The fusion polypeptide according to any one of claims 157-160, wherein at least one oligopeptide is operatively linked to the N-terminus of the modified TAF polypeptide, and at least one oligopeptide is operatively linked to the C-terminus of the modified TAF polypeptide.

163. The fusion polypeptide of claim 162, wherein the fusion polypeptide comprises two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the N-terminus of the modified TAF polypeptide, and/or two or more oligopeptides, three or more oligopeptides, four or more oligopeptides, five or more oligopeptides, six or more oligopeptides, eight or more oligopeptides, ten or more oligopeptides operably linked to the C-terminus of the modified TAF polypeptide.

164. The fusion polypeptide according to any one of claims 157-163, wherein the peptide bond comprises (i) methionine and the sequence-specific chemical cleavage is performed with cyanogen bromide, (ii) tryptophan and the sequence-specific chemical cleavage is performed with BNPS-skatole, (iii) an aspartic acid-proline (Asp-Pro) bond and the sequence-specific chemical cleavage is performed with formic acid, (iv) an asparagine-glycine bond and the sequence-specific chemical cleavage is performed with hydroxylamine, or (v) cysteine and the sequence-specific chemical cleavage is performed with NTCB.

165. The fusion polypeptide according to any one of claims 157-163, wherein the peptide bond comprises an Asp-Pro bond and the sequence-specific chemical cleavage is an acid cleavage.

166. The fusion polypeptide according to any one of claims 157-165, wherein the oligopeptides are operatively linked by the peptide bonds and are capable of being released from each other by the sequence-specific chemical cleavage.

167. The fusion polypeptide according to any one of claims 157-165, wherein the oligopeptides are operatively linked by different peptide bonds and are capable of being released from each other by different sequence-specific chemical cleavages.

168. The fusion polypeptide of claim 167, wherein the different peptide bonds comprise (i) methionine and the different sequence-specific chemical cleavage is performed with cyanogen bromide, (ii) tryptophan and the different sequence-specific chemical cleavage is performed with BNPS-skatole, (iii) aspartic acid-proline (Asp-Pro) bond and the different sequence-specific chemical cleavage is performed with formic acid, (iv) asparagine-glycine bond and the different sequence-specific chemical cleavage is performed with hydroxylamine, or (v) cysteine and the different sequence-specific chemical cleavage is performed with NTCB.

169. The fusion polypeptide of claim 167, wherein the different peptide bonds comprise an Asp-Pro bond and the different sequence-specific chemical cleavages are acid cleavages.

170. The fusion polypeptide according to any one of claims 157-169, wherein the oligopeptide is at least 4 amino acids long, at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long, at least 15 amino acids long, at least 20 amino acids long, or at least 25 amino acids long.

171. The fusion polypeptide according to any one of claims 157-170, wherein the oligopeptide is less than 50 amino acids long, less than 45 amino acids long, less than 40 amino acids long, less than 35 amino acids long, less than 30 amino acids long, less than 25 amino acids long, or less than 20 amino acids long.

172. The fusion polypeptide according to any one of claims 157-171, wherein the oligopeptide is 4 amino acids to 50 amino acids long, 6 amino acids to 40 amino acids long, 6 amino acids to 30 amino acids long, or 8 amino acids to 25 amino acids long.

173. A nucleic acid encoding a fusion polypeptide according to any one of claims 22-39, a modified RAF polypeptide according to claim 155 or claim 156, or a fusion polypeptide according to any one of claims 157-172.

174. A cell comprising the nucleic acid according to claim 173.

175. The cell of claim 174, wherein the cell is a bacterial cell or a yeast cell.

176. The cell of claim 174, wherein the bacteria is Escherichia coli or sodium-dependent Vibrio.

177. The cell of claim 176, wherein the cell is a BL21 bacterial cell.

178. The cell of claim 176, wherein the cell does not express Lon and ompT proteases.

179. The nucleic acid according to claim 173, wherein the nucleic acid is an isolated nucleic acid.