CN115717130B - Preparation method of aminoacyl-tRNA synthase mutant and alkenyl tyrosyl-tRNA - Google Patents

Abstract

The invention provides a preparation method of aminoacyl-tRNA (transfer ribonucleic acid) synthase mutant and alkenyl tyrosyl-tRNA. Wherein the aminoacyl-tRNA synthase mutant comprises: SEQ ID NO:1, and the mutation is selected from any one or more of the mutations of N160, A31, L32, L65, A67, L69, S107, F108, Q109, L110, P158, L159, Y161 and E162. Can specifically identify tyrosine derivatives, catalyze the combination of the tyrosine derivatives and corresponding tRNA to form alkenyl tyrosyl-tRNA, and is suitable for the field of enzyme catalysis.

Description

Preparation method of aminoacyl-tRNA synthase mutant and alkenyl tyrosyl-tRNA

Technical Field

The present invention relates to the field of enzyme catalysis, and in particular to a method for preparing an aminoacyl-tRNA synthase mutant and an alkenyl tyrosyl-tRNA.

Background Art

Allyl groups are widely used in organic synthesis. They can participate in metathesis, Diels-Alder reaction (dienes addition reaction), 1,3-dipolar cycloaddition reaction (1,3-Dipolar Cycloaddition), and have been used in amino acid derivative cross-linking reaction and cyclopeptide synthesis. Allyl-L-tyrosine (O-allyl-L-tyrosine, OAY) is an important non-natural amino acid. It is introduced into proteins or peptides and further reacts with other functional groups through chemical reactions, so as to achieve the purpose of protein labeling and protein-to-molecule coupling. The introduction of non-natural amino acids through orthogonal tRNA, aminoacyl-tRNA synthetase (aaRS) and amber codon (TAG) has been proven to be an efficient and feasible solution. However, since the non-natural amino acids introduced are mostly derivatives of natural amino acids, the specificity is not strong.

Summary of the invention

The main purpose of the present invention is to provide a method for preparing an aminoacyl-tRNA synthase mutant and an alkenyl tyrosyl-tRNA, so as to solve the problem of poor specificity of introducing tyrosine derivatives into proteins in the prior art.

In order to achieve the above object, according to the first aspect of the present invention, there is provided an aminoacyl-tRNA synthase mutant, comprising: SEQ ID A protein in which the amino acid sequence of pNFRS shown in NO: 1 is mutated, wherein the mutation is selected from the N160 mutation and any one or more of the following mutations: A31 is mutated to A31S, A31G, A31C, A31T or A31L; L32 is mutated to L32G, L32S, L32C, L32T, L32V, L32A or L32I; L65 is mutated to L65V, L65T, L65C or L65I; A67 is mutated to A67S; L69 is mutated to L69S, L69V or L69I; S107 is mutated to S107R, S107A or S107T; F108 is mutated to F108S, F108H or F108W; Q109 is mutated to Q109G, Q109S, Q109V or Q109N; L110 is mutated to L110R, L110Y, L110K, L110I or L110V; P158 mutated to P158A, P158L, P158S, P158D or P158M; L159 mutated to L159T, L159Q, L159E, L159V, L159D, L159I or L159A; Y161 mutated to Y161M, Y161Q , Y161N, Y161S, Y161W or Y161F; E162 mutates to E162V, E162M, E162L, E162T, E162I or E162D; N160 mutates to N160S, N160G, N160E, N160F, N160D, N160A, N160I, N160M, N160H or N160V.

Furthermore, the aminoacyl-tRNA synthase mutant is selected from a mutant having any of the following combined mutations based on the amino acid sequence of pNFRS shown in SEQ ID NO: 1,

1)A31S+L32G+P158A+L159T+N160S+Y161M+E162V;

2)A31G+L32S+L65V+L69S+S107R+F108S+Q109G+L110R+P158L+L159Q+N160G+Y161Q+E162M;

3)A31G+L32C+P158S+L159E+N160E+Y161N+E162L;

4)A31C+L32T+P158S+L159Q+N160F+Y161S+E162T;

5)A31G+L32V+L65T+A67S+S107A+F108H+Q109S+L110Y+P158D+L159V+N160D+Y161Q+E162I;

6)A31T+L32V+L65C+L69V+S107A+F108S+Q109V+L110K+P158M+L159T+N160G+Y161W+E162T;

7)L32S+L159D+N160A+Y161S+E162T;

8)N160H+L32V+S107T+Q109N+L110I+L159I;

9)N160H+L32A;

10)N160H+L32V;

11)N160H+L32I+L65V+A67S+S107T+F108W+L110R+P158A+L159D+E162D;

12)N160H+A31L+L32A+L65I+L69I+L110V+L159A+Y161F.

In order to achieve the above object, according to the second aspect of the present invention, a DNA molecule is provided, which encodes the above aminoacyl-tRNA synthase mutant.

In order to achieve the above object, according to the third aspect of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above DNA molecule.

In order to achieve the above object, according to the fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the above DNA molecule or the above recombinant plasmid.

Further, the host cell includes a prokaryotic cell; preferably, the prokaryotic cell includes Escherichia coli.

In order to achieve the above object, according to the fifth aspect of the present invention, a method for preparing alkenyl tyrosyl-tRNA is provided, the method comprising using the above aminoacyl-tRNA synthase mutant to catalyze the binding of alkenyl tyrosine and tRNA to prepare alkenyl tyrosyl-tRNA, wherein alkenyl tyrosine is a non-natural amino acid.

Further, alkenyl tyrosine includes allyl tyrosine, and further includes O-allyl-L-tyrosine.

Furthermore, the concentration of O-allyl-L-tyrosine is 1 to 5 mM.

Furthermore, the aminoacyl-tRNA synthase mutant contained in the above host cell is used to catalyze the binding of alkenyl tyrosine and tRNA; preferably, alkenyl tyrosine is dissolved in 2-10M sodium hydroxide to form an alkenyl tyrosine solution, and the pH of the alkenyl tyrosine solution is 9-11.

By applying the technical solution of the present invention and utilizing the aminoacyl-tRNA synthase mutant, tyrosine derivatives can be recognized with high specificity, and catalyze the binding of tyrosine derivatives with corresponding tRNA to form alkenyl tyrosyl-tRNA.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

FIG. 1 shows a statistical graph of fluorescence values per unit bacterial concentration corresponding to different aminoacyl-tRNA synthase mutants according to Example 8 of the present invention.

DETAILED DESCRIPTION

It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

Terminology explanation:

Unnatural amino acids: Unnatural amino acids refer to amino acids that are not encoded by the existing 64 genetic codons, that is, amino acids that are different from the existing 20 natural amino acids.

Orthogonality of the introduction of non-natural amino acids: Exogenously introduced aminoacyl-tRNA synthase (aaRS) only recognizes exogenously introduced tRNA, connects non-natural amino acids and tRNA to form a certain aminoacyl-tRNA, and cannot cross-react with endogenous aminoacyl-tRNA synthase, endogenous tRNA and natural amino acids in the host cell. A certain aminoacyl-tRNA is the product formed by the connection of a certain amino acid and tRNA, such as tyrosine and tRNA to form tyrosyl-tRNA.

As mentioned in the background art, the aminoacyl-tRNA synthetase used in the prior art can catalyze the binding of amino acid derivatives with corresponding tRNA to generate a certain aminoacyl-tRNA. However, the binding specificity for non-natural amino acids is not strong, which can easily lead to the introduction of natural amino acids in specific applications, resulting in impure products.

Therefore, in this application, for the same problems that exist in the introduction of tyrosine derivatives such as O-allyl-L-tyrosine (OAY), the inventors, on the one hand, constructed a saturated mutation library of 14 sites for the tyrosyl-tRNA synthase mutant pNFRS from Methanocaldococcus jannaschii, and obtained highly specific aminoacyl-tRNA synthase mutants after multiple rounds of screening; on the other hand, the inventors tried to use pNFRS-160H with lower natural amino acid introduction as a starting template, and obtained aminoacyl-tRNA synthase mutants with higher specificity after multiple rounds of screening through a positive and negative screening system based on red fluorescent protein and antibiotic resistance genes. Therefore, a series of protection schemes for this application are proposed.

In a first typical embodiment of the present application, an aminoacyl-tRNA synthase mutant is provided, comprising: a protein in which the amino acid sequence of pNFRS shown in SEQ ID NO: 1 is mutated, wherein the mutation is selected from N160 mutation and any one or more of the following mutations: A31 is mutated to A31S, A31G, A31C, A31T or A31L; L32 is mutated to L32G, L32S, L32C, L32T, L32V, L32A or L32I; L65 is mutated to L65V, L65T, L65C or L65I; A67 mutated to A67S; L69 mutated to L69S, L69V or L69I; S107 mutated to S107R, S107A or S107T; F108 mutated to F108S, F108H or F108W; Q109 mutated to Q109G, Q109S, Q109V or Q109N; L110 mutated to L110R, L110Y, L110K, L110I or L110V; P158 mutated to P158A, P158L, P158T, P158S, P158D or P158M; L159 mutated to L159T, L159Q, L159C, L159E, L159V, L159D, L159I or L159A; Y161 mutated to Y161M, Y161Q, Y161T, Y161N, Y161 61S, Y161W or Y161F; E162 mutates to E162V, E162M, E162A, E162L, E162T, E162I or E162D; wherein the above-mentioned N160 mutates to N160S, N160G, N160L, N160E, N160F, N160D, N160A, N160I, N160M, N160H or N160V.

The inventors compared the mutation of N160 to H160 or S207 to A207 based on pNFRS. After testing, it was found that the mutation of N160 to H160 had a lower introduction of natural amino acids. Based on structural analysis, it was found that N160 could form a hydrogen bond with the hydroxyl group of Tyr (tyrosine). When N160 mutated to H or other amino acids, the hydrogen bond was destroyed, resulting in the inability of the aminoacyl-tRNA synthase to form a hydrogen bond with Tyr. Therefore, it is speculated that pNFRS-160H is used as a starting template for the para-hydroxyl group of tyrosine. Removing the other 13 amino acids at position 160 to construct a multi-site saturation mutation library may result in more specific aminoacyl-tRNA synthase mutants. As a result, more efficient and specific aminoacyl-tRNA synthase mutants with the introduction of non-natural amino acids were indeed obtained through high-throughput screening.

In a preferred embodiment, the aminoacyl-tRNA synthase mutant is selected from a mutant having any of the following combined mutations based on the amino acid sequence of pNFRS shown in SEQ ID NO: 1:

1) OAYRS-1#: A31S+L32G+P158A+L159T+N160S+Y161M+E162V;

2) OAYRS-3#: A31G+L32S+L65V+L69S+S107R+F108S+Q109G+L110R+P158L+L159Q+N160G+Y161Q+E162M;

3) OAYRS-8#: A31G+L32C+P158S+L159E+N160E+Y161N+E162L;

4) OAYRS-10#: A31C+L32T+P158S+L159Q+N160F+Y161S+E162T;

5) OAYRS-12#: A31G+L32V+L65T+A67S+S107A+F108H+Q109S+L110Y+P158D+L159V+N160D+Y161Q+E162I;

6) OAYRS-14#: A31T+L32V+L65C+L69V+S107A+F108S+Q109V+L110K+P158M+L159T+N160G+Y161W+E162T;

7) OAYRS-18#: L32S+L159D+N160A+Y161S+E162T;

8) OAYRS-6#: N160H+L32V+S107T+Q109N+L110I+L159I;

9)OAYRS-13#:N160H+L32A;

10) OAYRS-25#: N160H+L32V;

11) OAYRS-30#: N160H+L32I+L65V+A67S+S107T+F108W+L110R+P158A+L159D+E162D;

12) OAYRS-35#: N160H+A31L+L32A+L65I+L69I+L110V+L159A+Y161F.

The above aminoacyl-tRNA synthase mutants are all mutants obtained by mutation based on the amino acid sequence of pNFRS. Compared with the original protein and the existing protein, the above aminoacyl-tRNA synthase mutants have higher specificity for the introduction of tyrosine derivatives.

The distance between the above mutation sites and the para-hydroxyl group of tyrosine is Therefore, the binding energy of the above site with the para-hydroxyl group of tyrosine is relatively large, which has a greater impact on the specific binding of tyrosine. Compared with the substrate tyrosine, derivatives of non-natural amino acid tyrosine (such as allyl tyrosine) can undergo substitution reactions at the position of the para-hydroxyl group of tyrosine, forming an ether bond instead of the hydroxyl group. Therefore, exploring the amino acids near this para-hydroxyl group can affect the binding specificity of the protein to the substrate.

But because There are too many sites within the protein structure and activity of multiple site mutations, and it is difficult to simulate them by computer speculation, and the results obtained by simulation also have large deviations in practical applications. Therefore, it is necessary to conduct a large number of screenings through actual experiments to obtain the specific combination of mutation sites that can produce aminoacyl-tRNA synthase mutants with better activity.

In a second typical embodiment of the present application, a DNA molecule is provided, which encodes the above-mentioned aminoacyl-tRNA synthase mutant.

In a third typical embodiment of the present application, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above-mentioned DNA molecule.

The above DNA can encode the above aminoacyl-tRNA synthase mutant, and can be connected to a recombinant plasmid to form a circular DNA. The above DNA and recombinant plasmid can be transcribed and translated under the action of RNA polymerase, ribosome, tRNA, etc. to obtain the above aminoacyl-tRNA synthase mutant. For different host species of DNA molecules or recombinant plasmids, the nucleotide sequence can be flexibly codon-optimized using existing technology to obtain a nucleotide sequence with higher transcription and translation efficiency.

In a fourth typical embodiment of the present application, a host cell is provided, wherein the host cell contains the above-mentioned DNA molecule or recombinant plasmid.

In a preferred embodiment, the host cell comprises a prokaryotic cell; preferably, the prokaryotic cell comprises Escherichia coli.

Utilize the above-mentioned host cell, can carry out the replication of recombinant plasmid in host cell, can also carry out transcription, translation of DNA molecule carried on recombinant plasmid, obtain a large amount of aminoacyl-tRNA synthase mutant.In the above-mentioned host cell, by introducing the expression plasmid of exogenous tRNA, host cell can synthesize exogenous tRNA corresponding to aminoacyl-tRNA, and express aminoacyl-tRNA synthase mutant, extra tyrosine derivative is added as substrate in the outside, namely can carry out catalysis for tyrosine derivative and tRNA in organism, obtain corresponding aminoacyl-tRNA.Due to the binding specificity of aminoacyl-tRNA synthase mutant, although natural amino acids such as tyrosine can be synthesized by itself in host cell, the tyrosine that can be combined with tRNA is less, namely the final generation is mainly a certain aminoacyl-tRNA produced by the combination of non-natural amino acid and tRNA, and the target non-natural amino acid is carried on this aminoacyl-tRNA.

In a fifth typical embodiment of the present application, a method for preparing alkenyl tyrosyl-tRNA is provided, using the above-mentioned aminoacyl-tRNA synthase mutant to catalyze the binding of alkenyl tyrosine and tRNA to prepare alkenyl tyrosyl-tRNA.

In a preferred embodiment, the alkenyl tyrosine comprises O-allyl-L-tyrosine.

In a preferred embodiment, the concentration of O-allyl-L-tyrosine is 1-5 mM; preferably, the aminoacyl-tRNA synthase mutant contained in the above host cell is used to catalyze the binding of alkenyl tyrosine and tRNA, and the binding has the orthogonality of the introduction of non-natural amino acids; preferably, alkenyl tyrosine is dissolved in 2-10 M sodium hydroxide to form an alkenyl tyrosine solution, and the pH of the alkenyl tyrosine solution is 9-11; more preferably, the concentration of sodium hydroxide is 10 M, and the pH of the alkenyl tyrosine solution is 10.

Utilize the above-mentioned preparation method, utilize aminoacyl-tRNA synthase mutant to catalyze O-allyl-L-tyrosine and other alkenyl tyrosines to combine with corresponding tRNA, thus prepare alkenyl tyrosyl-tRNA.tRNA and the corresponding amino acid to be introduced are theoretically one-to-one corresponding relationship.In order to achieve the purpose of introducing specific amino acids, it is necessary to ensure that the aminoacyl-tRNA synthase mutant, specific amino acids (i.e. non-natural amino acids) and corresponding tRNA adopted can not cross-react with the synthase, natural amino acids and tRNA in host cell (such as Escherichia coli) source.Therefore, the method can be used for the fixed-point introduction of non-natural amino acids in vivo on the one hand, and the preparation method can flexibly select enzyme catalysis, immobilization, biotransformation and other modes to carry out the in vitro preparation of alkenyl tyrosyl-tRNA in addition, so as to be used for adopting the mode of cell-free synthesis to carry out the efficient synthesis of non-natural amino acid protein.

Based on the host cell's ability to produce tRNA raw materials by itself, the host cell carrying the above-mentioned DNA molecule, exogenous orthogonal tRNA, and recombinant plasmid can express exogenous aminoacyl-tRNA synthase mutants and exogenous orthogonal tRNA, thereby exerting a catalytic effect to obtain alkenyl tyrosyl-tRNA products. The catalytic reaction is a bioorthogonal reaction, and the compounds produced in the host cell, including natural amino acids, do not participate in the catalytic reaction; the alkenyl tyrosine, exogenous orthogonal tyrosine tRNA, and alkenyl tyrosyl-tRNA products required for the catalytic reaction do not affect the host cell's own biochemical reactions and life activities.

The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

Example 1 Construction of a screening scheme based on red fluorescent protein and chloramphenicol resistance

A screening scheme based on red fluorescent protein and chloramphenicol resistance was constructed, specifically using the coding gene of red fluorescent protein mcherry to replace the green fluorescent protein gene GFPuv in the screening plasmid in the literature. The mcherry sequence with codon optimization was synthesized by Jin Weizhi, and the NcoI restriction site was introduced at the 5' end of the sequence, and the XhoI restriction site was introduced at the 3' end of the sequence, and constructed in the pET-28a vector. The sequence is shown in SEQ ID NO: 3.

SEQ ID NO: 3: ccatgggcatgggcgttagcaaaggcgaagaagataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggcagcgtgaacggccatgagtttgaaatcgagggcgaaggcgaaggtcgcccatacgaaggcacccagaccgccaaactgaaagtgacgaaaggcggtccgctg ccattcgcgtgggatattctgagcccacagttcatgtacggcagcaaagcctacgtgaagcatccggccgatatcccggattatctgaagctgagcttcccagagggcttcaagtggggagcgcgtgatgaactttgaagatggcggtgtggttaccgttacgcaagatagcagtc tgcaagatggcgagttcatctacaaggttaagctccgcggcaccaacttcccgagcgatggtccggtgatgcagaaaaagacgatgggctgggaagcgagcagcgaacgcatgtatccagaagatggcgcgctgaaaggcgagatcaaacagcgtctgaagctgaaagacggcggccattacgatgcggaggtgaaga ccacctacaaggcgaaaaagccggttcagctgccgggcgcgtacaacgtgaacatcaagctggacatcaccagccacaacgaagactacaccatcgtggagcagtacgaacgcgcggaaggtcgccatagtaccggcggcatggacgaactgtataaataatgactcgag.

The expression plasmid was transformed into BL21 (DE3) competent cells, and the solid LB medium plates containing LB + 50 μg/mL kanamycin were coated. After overnight culture, a single clone was obtained. The single clone was inoculated into 5 ml LB medium and cultured at 37°C with shaking at 200 rpm for 2-3 h. When the _OD600 was 0.6-0.8, a final concentration of 0.5 mM IPTG was added and induced at 30°C for 20 h. The culture solution turned purple-red, indicating that the red fluorescent protein was available. The sequences of the green fluorescence and antibiotic screening plasmid except the green fluorescent protein coding sequence were amplified by PCR using RFP-HR-Bone-F (SEQ ID NO: 4: gagtcgtattaatttcgcgggatcgagtgagcgcaacgcaattaatg) and RFP-HR-Bone-R (SEQ ID NO: 5: gcattaagcgcggcgggtgtggtgttttcaccgtcatcaccg) as primers (N at Biotechnol. 2002 Oct; 20(10): 1044-8.), and the sequences of the green fluorescence and antibiotic screening plasmid except the green fluorescent protein coding sequence were amplified by PCR using RFP-HR-F: (SEQ ID NO: 6: cattaattgcgttgcgctcactcgatcccgcgaaattaatacgactc) and RFP-HR-R: (SEQ ID NO: 7: cattaattgcgttgcgctcactcgatcccgcgaaattaatacgactc) as primers. NO:7:cggtgatgacggtgaaaacaccacacccgccgcgcttaatgc) was used as primers, PCR was used to amplify the mcherry coding frame sequence, including the T7 promoter and T7 terminator, and then a plasmid was constructed through homologous recombination, thus completing the replacement of the fluorescent protein. The constructed screening plasmid was transformed into DH10B competent cells, and after sequencing, a DH10B strain containing the screening plasmid was obtained, named DH10B-REP (screening plasmid), and prepared into an electroporation competent cell for standby use.

Example 2 Construction of pET-Gln constitutive expression plasmid

In order to achieve constitutive expression of aminoacyl-tRNA synthase, the promoter and terminator on pET-28a were replaced with Gln promoter and GlnTT terminator. The promoter and terminator were synthesized into a DNA sequence by GeneWeizhi. A BglII restriction site was introduced at the 5' end of the promoter, NdeI and EcoRI sites were introduced at the 3' end, and an XhoI site was introduced at the 3' end of the GlnTT terminator. The synthesized sequence was ligated to the pET28a vector by BglII and XhoI restriction enzymes, and pET-Gln was obtained after sequencing for later use.

The synthetic promoter and terminator sequences are shown in SEQ ID NO:8.

SEQ ID NO: 8: agatctgagctcccggtcatcaatcatccccataatccttgttagatgatcaattttaaaaaactaacagttcagcctgtcccgcttataagatccgttatacgtttacgctttgaggaatcccatcatatggaattcctgcagtttcaaacgctaaattgcctgatgcgctacgcttatcaggcctacat gatctctgatatattgagtacgtcttttg taggccggataatcgttcactcgcatccggcagaaacagcaacatccaaaacgccgcgttcagcggcgtttatgctttcttcgcgaattaattccgcttcgcaacatgtgagcaccggtttattgactaccggaagcagtgtgaccgtgtgctttaaatgcctgaggccagtttgctcaggctctccccgtgg aggtaataattgacgatatgatcactcgag.

Example 3 Construction of pNFRS synthase mutant library

The amino acid sequence of pNFRS is shown in SEQ ID NO:1.

SEQ ID NO: 1:

MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSSFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNPLNYEGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGK MSSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL.

The nucleotide sequence encoding pNFRS is shown in SEQ ID NO:2.

SEQ ID NO 2: atggatgaatttgaaatgattaaacgcaacaccagcgaaattattagcgaagaagaactgcgcgaagtgctgaagaaggacgagaagtcagctctgattggctttgaaccgagcggcaagatacacctgggccattatttacagattaagaagaagatgatcgatttacagaacgcgggctttgatattattattctgctggcggatctgcatg cgtatctgaaccagaaag gcgaactggatgaaattcgcaagatcggagattataacaagaaggtatttgaggcgatgggcctgaaagcgaaatatgtgtatggcagcagctttcagctggataaagattataccctgaacgtgtatcgcctggcgctgaagacgacactgaaacgcgcgcgccgcagcatggaactgattgcgcgcgaagatgagaat cccaaagtggcggaagtgatttatccgatta tgcaggtgaacccgctgaactatgaaggcgtggatgtggcggtgggcggcatggaacagcgcaagatacacatgctggcgcgcgaactgctgccgaagaaggtagtttgcattcataacccggtgctgaccggcctggatggcgaaggcaagatgtccagcagcaaaggcaactttattgcggtggatgatagcccggaagaaatt cgcgcgaagatcaagaaagcgtact gcccggcgggcgtggtggaaggcaacccgattatggaaattgcgaaatatttcttagagtatccgctgaccattaaacgcccggagaagttcggtggcgatctgaccgtgaacagctatgaagaactggaaagcctgtttaagaataaggaactgcatccgatggatctgaagaatgctgtggcggaagaactgattaagatactcgaaccgattcg caaacgcctgtaa.

Molecular docking was used to find the distance between the para-hydroxyl group of pNFRS and tyrosine Multiple-point saturation mutagenesis was performed on the amino acids within (31, 32, 65, 67, 69, 107, 108, 109, 110, 158, 159, 160, 161, 162). Primers used:

31-32NNK, SEQ ID NO: 9: gctgaagaaggacgagaagtcannknnkattggctttgaaccgagcggcaagatac.

65-69-MNN, SEQ ID NO: 10: tctggttcagatacgcatgmnnatcmnncagmnnaataataatatcaaagcccgcgttctg.

65-69NNK, SEQ ID NO: 11: gggctttgatattattattnnkctgnnkgatnnkcatgcgtatctgaaccagaaaggcgaactg.

107-110MNN, SEQ ID NO: 12: cacgttcagggtataatctttatcmnnmnnmnnmnngctgccatacacatatttcgctttcaggcccatc.

107-110NNK, SEQ ID NO: 13: gcgaaatatgtgtatggcagcnnknnknnknnkgataaagattataccctgaacgtgtatcgcctg.

158-162MNN, SEQ ID NO: 14: catgccgcccaccgccacatccacgccmnnmnnmnnmnnmnngttcacctgcataatcggataaatcacttccgc.

The primers of the present application contain degenerate bases, wherein the degenerate base n represents any one of a, t, g, and c, the degenerate base k represents g or t, and the degenerate base m represents a or c.

The above primers were used to amplify segments 31-69, 65-110, and 107-162, respectively, and then the NNK-containing fragment of pNFRS (31-162aa) was obtained by over-lap PCR. The vector part was obtained by PCR amplification using SEQ ID NO: 15: tgacttctcgtccttcttcagcacttcg (31-bone-R) and SEQ ID NO: 16: ggatgtggcggtgggcggcatg (162-bone-F) as primers. The described part is placed here. The NNK fragment and the vector part are connected by circular polymerase extension cloning (CPEC). The construction steps are as follows: PCR amplification of the first fragment with 31-32NNK and 65-69NNK as primers, PCR amplification of the second fragment with 65-69NNK and 107-110MNN as primers, PCR amplification of the third fragment with 107-110NNK and 158-162MNN as primers, and then overlap PCR amplification of the fused NNK fragment with 31-32NNK and 158-162MNN as primers, and then the fusion fragment is connected to the plasmid backbone by the CPEC method. The recombinant vector is transformed into DH10B electroporation competent cells by electroporation, and kanamycin is added for overnight culture. The mutant mixed plasmid is extracted using a plasmid extraction kit and stored at -20°C for use.

Example 4 Selection of mutation template

In order to improve the specific introduction of non-natural amino acids and improve the efficiency of screening, reducing or eliminating the introduction of natural amino acids by aminoacyl-tRNA synthase will help the screening process. Initially, the wild-type aminoacyl-tRNA synthase MjTyrRS from Methanocaldococcusjannaschii was used for screening. Due to its specific recognition of the natural substrate tyrosine Tyr, the success rate of screening was not high, and the specificity of the mutations obtained by screening was poor. Therefore, we speculated that the use of low-background MjTyrRS mutants would help obtain efficient and specific novel aminoacyl-tRNA synthase mutants. Combined with literature research, it was found that the MjTyrRS mutant pNFRS can introduce p-nitro-L-phenylalanine pNF, p-iodo-L-phenylalanine pIF and tyrosine, but with poor specificity. Therefore, a codon-optimized pNFRS sequence was synthesized. Based on the above examples, combined with structural simulation and docking analysis, further mutations were performed to obtain N160H (nucleotides 478-480, original nucleotides aac, mutated to cat encoding His) and S207A (nucleotides 619-621 agc, mutated to gcg encoding alanine), and amino acid introduction was compared to find a more suitable template for mutation studies.

The coding sequences of pNFRS, pNFRS-160H and pNFRS-S207A were connected to pET-Gln, respectively, and transformed into DH10B together with the red fluorescent protein-based screening plasmid REP. They were cultured in LB medium with or without the corresponding amino acids. The results are shown in Table 1.

Table 1

Therefore, the optimal initial template is pNFRS-160H. In order to verify the feasibility of our method, we selected pNFRS and pNFRS-160H as templates, respectively, constructed a multi-point mutation library, and performed screening.

Example 5 Construction of pNFRS-160H synthase mutant library

From the conclusions obtained from the experiment in Example 4, it was found that after the 160H mutation, the specificity of introducing non-natural amino acids was enhanced, so a synthase mutant library was constructed, retaining the 160H site, and the other primers used were the same as in Example 3, except that the primers 158-162MNN were changed, as shown in SEQ ID NO: 17.

SEQ ID NO: 17: catgccgcccaccgccacatccacgccmnnmnnatgmnnmnngttcacctgcataatcggataaatcacttccgc.

Similarly, three short fragments were amplified by PCR, and the NNK-containing fragment of pNFRS-160H (31-162aa) was further obtained by over-lap PCR and connected to the vector CPEC method, and electroporated DH10B electroporation competent cells, kanamycin was added for overnight culture, and the plasmid was extracted and stored at -20°C for future use.

Example 6 Screening of synthase mutants from pNFRS and pNFRS-160H mutant libraries

The synthase mutants obtained in Examples 3 and 5 were electro-transformed into DH10B-REP, and after resuscitation at 37°C for 1 h, they were spread on LB solid screening plates (hereinafter referred to as B plates) containing 10 μg/mL tetracycline, 50 μg/mL kanamycin, 50 μg/mL chloramphenicol, 0.1% arabinose, and 1 mM OAY for culture. The plates were cultured for 48 to 72 h until red clones grew out. The red clones were plated on plates B and C (the 1 mM OAY on the B plate was removed). OAY), and clones that did not grow on C plate but grew on B plate and had red fluorescence were selected and streaked on B plate. After red clones grew out, 2 to 3 rounds of screening were further performed on B and C plates. As a result, 1#, 3#, 8#, 10#, 12#, 14# and 18# synthase mutants were screened from the pNFRS mutation library and introduced into OAY. 6#, 13#, 25#, 30# and 35# synthase mutants were screened from the pNFRS-160H mutation library. The synthase mutants were further sequenced, and the results are shown in Table 2.

Table 2

The red clones were cultured in shake flasks and compared. -OAY was the experimental group without the addition of unnatural amino acids, and OAY corresponded to the addition of 1 mM OAY. The _OD600 value was measured at the same time to calculate the fluorescence ratio per unit biomass. The results are shown in Table 3.

Table 3

Example 7 Construction of pNFRS and pNFRS-160H mutants and tRNA co-expression plasmids

tRNA-XhoI-F (SEQ ID NO: 18: gggctcgagcccatcaaaaaaatattctcaac) and tRNA-HR-XhoI-R (SEQ ID NO: 19: gggctcgagcccatcaaaaaaatattctcaac) were used. NO: 19: gggctcgagtaaaaaaaatccttagctttc), the coding sequence of TyrT, including the promoter and terminator, was amplified from the screening plasmid REP, the product was digested with XhoI and connected with pET-Gln-pNFRS mutants (1#, 3#, 7#, 8#, 10#, 12#, 14#, 18#), pET-Gln-pNFRS-160H mutants (6#, 13#, 25#, 30#, 35#), and sequencing was performed to obtain the correct plasmids pET-Gln-pNFRS (1#, 3#, 8#, 10#, 12#, 14# or 18#)-tRNA and pET-Gln-pNFRS-160H mutants (6#, 13#, 25#, 30# or 35#)-tRNA.

Example 8 Evaluation of Allyl-L-Tyrosine Introduction

The codon-optimized green fluorescent protein sfGFP gene was synthesized by GENEWIZ and constructed at the NcoI and XhoI sites of pACYCduet1. The sfGFP gene sequence is shown in SEQ ID NO: 20.

SEQ ID NO: 20: atgagcaaaggtgaagaactgtttaccggcgttgtgccgattctggtggaactggatggcgatgtgaacggtcacaaattcagcgtgcgtggtgaaggtgaaggcgatgccacgattggcaaactgacgctgaaatttatctgcaccaccggcaaactgccggtgccgtggccgacgctggtga ccaccctgacctatggcgttcagtgttttagtcgctatccggatcacatgaaacgtcacgatttctttaaatctgcaatgccggaaggctatgtgcaggaacgtacgattagctttaaagatgatggcaaatataaaacgcgcgccgttgtgaaatttgaaggcgataccctg gtgaaccgcattgaactgaaaggcacggattttaaagaagatggcaatatcctgggccataaactggaatacaactttaatagccataatgtttatattacggcggataaacagaaaaatggcatcaaagcgaattttaccgttcgccataacgttgaagatggcagtgtgcagctggcagatcattatcagcagaataccccgattggtgatggtccggt gctgctgccggataatcattatctgagcacgcagaccgttctgtctaaagatccgaacgaaaaaggcacgcgggaccacatggttctgcacgaatatgtgaatgcggcaggtattacgtggagccatccgcagttcgaaaaa.

In order to introduce non-natural amino acids into specific sites in sfGFP, the 3-codon encoding I39 was mutated from att to tag, and the pACYCduet-sfGFP (I39) plasmid was obtained by DNA sequencing.

Combine pET-Gln-pNFRS(1#)-tRNA, pET-Gln-pNFRS(3#)-tRNA, pET-Gln-pNFRS(8#)-tRNA, pET-Gln-pNFRS(10#)-tRNA, pET -Gln-pNFRS(12#)-tRNA, pET-Gln-pNFRS(14#)-tRNA, pET-Gln-pNFRS(18#)-tRNA, pET-Gln-pNFRS-160H(6#)-tRNA, pET - Gln-pNFRS-160H(13#)-tRNA, pET-Gln-pNFRS-160H(25#)-tRNA, pET-Gln-pNFRS-160H(30#)-tRNA, pET-Gln-pNFRS-160H(35#)-tRNA )-tRNA and pACYCduet-sfGFP(I39TAG) were co-transformed into BL21(DE3) competent cells, spread on LB culture plates containing kanamycin (kan) and chloramphenicol (Cm), and cultured at 37°C until the cells grew. A single clone. A single clone was selected and inoculated in 30 mL LB (kan + Cm + 1 mM OAY), and LB (kan + Cm) without OAY was used as a negative control. After induction with 1 mM IPTG at 30°C overnight, 485 nm was used as the excitation light, and 525 nm was used as the emission light. The fluorescence intensity of green fluorescent protein was measured, and OD ₆₀₀ was measured at the same time, and the fluorescence value per unit bacterial concentration was calculated. and the mutant synthase OAYRS-CK1 (32S, 107T, 158T, 159Y, 162A) reported in the literature (Santoro SW, Wang L, Herberich B, et al. An efficient system for the evolution of aminoacyl-tRNA synthetase specificity[J]. Nature Biotechnology, 2002, 20(10):1044-1048.) and the OAYRS-CKB (32Y, 107A, 158C, 159A, 162L) designed in this experiment were compared, and the results are shown in Table 4 and Figure 1. : 1, and the enzyme with N160H mutation based on the amino acid sequence of pNFRS, cannot introduce OAY. From the results, the new mutant carrying N160H has higher specificity for the introduction of unnatural amino acids.

Table 4

From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the above aminoacyl-tRNA synthase mutant can be used to recognize alkenyl tyrosine with high specificity, catalyze the binding of alkenyl tyrosine with the corresponding tRNA, and form alkenyl tyrosyl-tRNA, which can be applied to the field of site-specific introduction of non-natural amino acids in proteins or polypeptides and derived protein labeling, enzyme catalysis, protein and other molecular coupling.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (8)

1. A method for preparing an alkenyl tyrosyl-tRNA, characterized in that the alkenyl tyrosyl-tRNA is prepared by using an aminoacyl-tRNA synthase mutant to catalyze the binding of alkenyl tyrosine and tRNA, The alkenyl tyrosine is a non-natural amino acid; The aminoacyl-tRNA synthase mutant is selected from a mutant having any of the following combined mutations based on the amino acid sequence of pNFRS shown in SEQ ID NO: 1: 1) A31S+L32G+P158A+L159T+N160S+Y161M+E162V; 2) A31G+L32S+L65V+L69S+S107R+F108S+Q109G+L110R+P158L+L159Q+N160G+Y161Q+E162M; 3) A31G+L32C+P158S+L159E+N160E+Y161N+E162L; 4) A31C+L32T+P158S+L159Q+N160F+Y161S+E162T; 5) A31G+L32V+L65T+A67S+S107A+F108H+Q109S+L110Y+P158D+L159V+N160D+Y161Q+E162I; 6) A31T+L32V+L65C+L69V+S107A+F108S+Q109V+L110K+P158M+L159T+N160G+Y161W+E162T; 7) L32S+L159D+N160A+Y161S+E162T; 8) N160H+L32V+S107T+Q109N+L110I+L159I; 9) N160H+L32A; 10) N160H+L32V; 11) N160H+L32I+L65V+A67S+S107T+F108W+L110R+P158A+L159D+E162D; 12) N160H+A31L+L32A+L65I+L69I+L110V+L159A+Y161F; The alkenyl tyrosine is O-allyl-L-tyrosine. 2. The preparation method according to claim 1, wherein the concentration of the O-allyl-L-tyrosine is 1 to 5 mM. 3. The preparation method according to claim 1, wherein the alkenyl tyrosine is dissolved in 2-10 M sodium hydroxide to form an alkenyl tyrosine solution, and the pH of the alkenyl tyrosine solution is 9-11. 4. A DNA molecule, characterized in that the DNA molecule encodes the aminoacyl-tRNA synthase mutant in the preparation method of alkenyl tyrosyl-tRNA according to claim 1. 5. A recombinant plasmid, characterized in that the recombinant plasmid is connected to the DNA molecule according to claim 4. 6. A host cell, characterized in that the host cell contains the DNA molecule according to claim 4 or the recombinant plasmid according to claim 5; the host cell is not an animal or plant species. The host cell according to claim 6 , wherein the host cell comprises a prokaryotic cell. 8. The host cell according to claim 7, wherein the prokaryotic cell comprises Escherichia coli.