CN115717130A - aminoacyl-tRNA (tRNA) synthase mutant and preparation method of alkenyltyrosyl-tRNA - Google Patents
aminoacyl-tRNA (tRNA) synthase mutant and preparation method of alkenyltyrosyl-tRNA Download PDFInfo
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Abstract
The invention provides an aminoacyl-tRNA (transfer ribonucleic acid) synthase mutant and a preparation method of alkenyl tyrosyl-tRNA. Wherein the aminoacyl-tRNA synthase mutant comprises: SEQ ID NO:1, wherein the mutation is selected from N160 mutation and any one or more of A31, L32, L65, A67, L69, S107, F108, Q109, L110, P158, L159, Y161 and E162. Can recognize tyrosine derivatives with high specificity, 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
Technical Field
The invention relates to the field of enzyme catalysis, in particular to an aminoacyl-tRNA (tRNA) synthase mutant and a preparation method of alkenyl tyrosyl-tRNA.
Background
Allyl groups have found widespread use in organic synthesis, and can participate in metathesis, diels-Alder reaction (diene addition reaction), 1,3-Dipolar Cycloaddition reaction (1, 3-Dipolar Cycloaddition reaction), and have been used in amino acid derivative crosslinking reactions and cyclic peptide synthesis. allyl-L-tyrosine (OAY) is an important unnatural amino acid, is introduced into protein or polypeptide, and further reacts with other functional groups through chemical reaction, so that the purposes of protein labeling and protein and other molecule coupling can be realized. The introduction of unnatural amino acids via orthogonal trnas, aminoacyl-tRNA synthetases (aaRS) and amber codons (TAG) has proven to be an efficient and feasible solution, however, the specificity is not strong because the introduced unnatural amino acids are mostly derivatives of natural amino acids.
Disclosure of Invention
The invention mainly aims to provide an aminoacyl-tRNA (aminoacyl-tRNA) synthase mutant and a preparation method of alkenyl tyrosyl-tRNA, so as to solve the problem of poor specificity of protein introduced tyrosine derivative in the prior art.
To achieve the above object, according to a first aspect of the present invention, there is provided an aminoacyl-tRNA synthase mutant comprising: SEQ ID NO:1, and the mutation is selected from N160 mutation and any one or more of the following mutations: the A31 mutation is A31S, A31G, A31C, A31T or A31L; the L32 mutation is L32G, L32S, L32C, L32T, L32V, L32A or L32I; the L65 mutation is L65V, L65T, L65C or L65I; the A67 mutation is A67S; l69 is mutated into L69S, L69V or L69I; the S107 mutation is 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; the P158 is mutated into P158A, P158L, P158S, P158D or P158M; l159 is mutated to L159T, L159Q, L159E, L159V, L159D, L159I or L159A; y161 is mutated to Y161M, Y161Q, Y161N, Y161S, Y161W or Y161F; e162 mutations are E162V, E162M, E162L, E162T, E162I or E162D; the N160 mutation is N160S, N160G, N160E, N160F, N160D, N160A, N160I, N160M, N160H or N160V.
Further, the aminoacyl-tRNA synthase mutant is selected from the group consisting of SEQ ID NOs: 1, or a mutant which has any one of the following combinatorial mutations based on the amino acid sequence of pNFRS shown in 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。
to achieve the above object, according to a second aspect of the present invention, there is provided a DNA molecule encoding the aminoacyl-tRNA synthase mutant.
In order to achieve the above object, according to a third aspect of the present invention, there is provided a recombinant plasmid having the above DNA molecule ligated thereto.
In order to achieve the above object, according to a fourth aspect of the present invention, there is provided a host cell containing the above DNA molecule, or the above recombinant plasmid.
Further, host cells include prokaryotic cells; preferably, the prokaryotic cell comprises E.coli.
In order to achieve the above object, according to a fifth aspect of the present invention, there is provided a method for producing an alkenyltyrosyl-tRNA, the method comprising producing the alkenyltyrosyl-tRNA by catalyzing the binding between an alkenyltyrosine, which is an unnatural amino acid, and a tRNA using the aminoacyl-tRNA synthase mutant.
Further, alkenyltyrosines include allyltyrosine, further including O-allyl-L-tyrosine.
Further, the concentration of O-allyl-L-tyrosine is 1 to 5mM.
Further, the aminoacyl-tRNA synthase mutant contained in the host cell is used to catalyze the binding of alkenyltyrosine and tRNA; preferably, the 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 scheme of the invention, the aminoacyl-tRNA synthetase mutant can recognize the tyrosine derivative with high specificity, catalyze the combination of the tyrosine derivative and the corresponding tRNA and form the alkenyl tyrosyl-tRNA.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a graph showing a statistical plot of unit bacterial concentration fluorescence values for different aminoacyl-tRNA synthase mutants according to example 8 of the invention.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
Interpretation of terms:
unnatural amino acids: an unnatural amino acid refers to an amino acid that is not encoded by the existing 64 genetic codons, i.e., an amino acid that differs from the existing 20 natural amino acids.
Orthogonality of unnatural amino acid introduction: exogenously introduced aminoacyl-tRNA synthases (aaRS) recognize only exogenously introduced trnas, linking unnatural amino acids to trnas to form certain aminoacyl-trnas, and do not cross-react with aminoacyl-tRNA synthases endogenous to the host cell, endogenous trnas, and natural amino acids. A aminoacyl-tRNA is a product formed by linking an amino acid to a tRNA, e.g., tyrosine, to a tRNA.
As mentioned in the background, aminoacyl-tRNA synthetases used in the prior art catalyze the binding of amino acid derivatives to the corresponding tRNA to produce an aminoacyl-tRNA. However, the binding specificity of the non-natural amino acid is not strong, and the natural amino acid is easily introduced in the specific application process, so that the product is not pure.
Therefore, in the present application, aiming at the problems of the introduction of tyrosine derivatives such as O-allyl-L-tyrosine (OAY), the inventors constructed a saturated mutation library with 14 sites for tyrosyl-tRNA synthase mutant pNFRS derived from Methanodococcus jannaschii on the one hand, and obtained high specificity aminoacyl-tRNA synthase mutant through multiple rounds of screening; on the other hand, the inventor tries to obtain an aminoacyl-tRNA synthase mutant with higher specificity through multiple rounds of screening by using pNFRS-160H with lower natural amino acid introduction as a starting template and a positive and negative screening system based on red fluorescent protein and antibiotic resistance genes. Thus a series of protection schemes of the present application are proposed.
In a first exemplary embodiment of the present application, there is provided an aminoacyl-tRNA synthase mutant comprising: SEQ ID NO:1, and the mutation is selected from N160 mutation and any one or more of the following mutations: the A31 mutation is A31S, A31G, A31C, A31T or A31L; the L32 mutation is L32G, L32S, L32C, L32T, L32V, L32A or L32I; the L65 mutation is L65V, L65T, L65C or L65I; the A67 mutation is A67S; l69 is mutated into 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; the P158 is mutated into P158A, P158L, P158T, P158S, P158D or P158M; l159 is mutated to L159T, L159Q, L159C, L159E, L159V, L159D, L159I or L159A; y161 is mutated to Y161M, Y161Q, Y161T, Y161N, Y161S, Y161W or Y161F; e162 mutation to E162V, E162M, E162A, E162L, E162T, E162I or E162D; wherein the N160 mutation is N160S, N160G, N160L, N160E, N160F, N160D, N160A, N160I, N160M, N160H or N160V.
The inventor compares that mutation N160 is H160 on the basis of pNFRS, or mutation S207 is A207, tests show that the mutation of N160 into H160 has lower natural amino acid introduction, structural analysis shows that N160 can form hydrogen bond with hydroxyl of Tyr (tyrosine), and the hydrogen bond is destroyed after the mutation of N160 into H and other amino acids, so that the aminoacyl-tRNA synthase can not form hydrogen bond with Tyr, therefore, the pNFRS-160H is supposed to be used as a starting template to form hydrogen bond with tyrosine para-hydroxyl
Constructing a multi-site saturated mutation library by removing other 13 amino acids at position 160 in the library can obtain more specific aminoacyl-tRNA synthase mutants, and the result indeed obtains more efficient and specific aminoacyl-tRNA synthase mutants introduced by unnatural amino acids through high-throughput screening.
In a preferred embodiment, the aminoacyl-tRNA synthase mutant is selected from the group consisting of SEQ ID NOs: 1, and a mutant which has any one of the following combined mutations based on the amino acid sequence of pNFRS shown in the specification:
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 aminoacyl-tRNA synthase mutants are all mutants obtained by mutation based on the amino acid sequence of pNFRS, and have higher introduction specificity for tyrosine derivatives compared with the original protein and the existing protein.
The distances between the mutant sites and tyrosine para-hydroxyl are all between
Therefore, the binding energy of the site and the para-hydroxyl of tyrosine is large, and the specific binding effect on tyrosine is large. As a derivative of the unnatural amino acid tyrosine (e.g., allyltyrosine) as compared to the substrate tyrosine, substitution reaction can occur at the position of the hydroxyl group para to tyrosine to form an ether bond instead of the hydroxyl group. Therefore, the research on the amino acid near the para-hydroxyl can influence the binding specificity of the protein to the substrate.
But because of
Too many sites within it, it is difficult to simulate the structure and activity of a protein having multiple site mutations by a method such as computer estimation, and the simulation resultsThe results of (a) also show a large deviation in practical use. Therefore, it is necessary to obtain a large number of screens through practical tests to obtain a combination of specific mutation sites that will result in a better active aminoacyl-tRNA synthase mutant.
In a second exemplary embodiment of the present application, a DNA molecule is provided that encodes the aminoacyl-tRNA synthase mutant described above.
In a third exemplary embodiment of the present application, a recombinant plasmid is provided, to which the above-described DNA molecule is ligated.
The DNA encodes the aminoacyl-tRNA synthase mutant and can be connected to a recombinant plasmid to form a circular DNA. The aminoacyl-tRNA synthase mutant can be obtained by transcribing and translating the DNA and the recombinant plasmid under the action of RNA polymerase, ribosome, tRNA and the like. Aiming at different host types of DNA molecules or recombinant plasmids, the prior art can be utilized to flexibly perform codon optimization on the nucleotide sequence, thereby obtaining the nucleotide sequence with higher transcription and translation efficiency.
In a fourth exemplary embodiment of the present application, a host cell is provided, which 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 E.coli.
The host cell can be used to replicate the recombinant plasmid in the host cell, and can also be used to transcribe and translate the DNA molecule carried on the recombinant plasmid to obtain a large number of aminoacyl-tRNA synthase mutants. The host cell is enabled to synthesize the exogenous tRNA corresponding to the aminoacyl-tRNA by introducing an expression plasmid of the exogenous tRNA into the host cell, express an aminoacyl-tRNA synthase mutant, and add a tyrosine derivative as a substrate outside, namely, catalysis aiming at the tyrosine derivative and the tRNA can be carried out in an organism to obtain a corresponding aminoacyl-tRNA. Although the host cell can synthesize natural amino acids such as tyrosine by itself due to the binding specificity of the aminoacyl-tRNA synthase mutant, the host cell can synthesize fewer natural amino acids, that is, the aminoacyl-tRNA which is generated by combining the unnatural amino acid and the tRNA is mainly generated, and the aminoacyl-tRNA carries the target unnatural amino acid.
In a fifth exemplary embodiment of the present application, a method for producing an alkenyltyrosyl-tRNA is provided, wherein the aminoacyl-tRNA synthase mutant is used to catalyze the binding of alkenyltyrosine and tRNA to produce the alkenyltyrosyl-tRNA.
In a preferred embodiment, the alkenyltyrosine comprises O-allyl-L-tyrosine.
In a preferred embodiment, the concentration of O-allyl-L-tyrosine is 1 to 5mM; preferably, the aminoacyl-tRNA synthase mutant contained in the host cell is used for catalyzing the combination of alkenyl tyrosine and tRNA, and the combination has the orthogonality introduced by unnatural amino acid; preferably, the 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; more preferably, the sodium hydroxide concentration is 10M and the pH of the alkenyl tyrosine solution is 10.
The alkenyltyrosyl-tRNA is prepared by catalyzing the binding of an alkenyltyrosine, such as O-allyl-L-tyrosine, to the corresponding tRNA using an aminoacyl-tRNA synthase mutant according to the above-described preparation method. the tRNA theoretically corresponds to the amino acid to be introduced. For the purpose of introducing a specific amino acid, it is necessary to ensure that the aminoacyl-tRNA synthase mutant, the specific amino acid (i.e., the unnatural amino acid), and the corresponding tRNA used do not cross-react with the host cell (e.g., E.coli) derived synthase, the natural amino acid, and the tRNA. Therefore, on one hand, the method can be used for the site-specific introduction of the unnatural amino acid in vivo, and in addition, the preparation method can flexibly select enzyme catalysis, immobilization, biotransformation and other modes for the in vitro preparation of the alkenyl tyrosyl-tRNA, so that the method can be used for the efficient synthesis of the protein containing the unnatural amino acid in a cell-free synthesis mode.
Based on the fact that the host cell can generate tRNA raw materials by itself, the host cell carrying the DNA molecules, the exogenous orthogonal tRNA and the recombinant plasmid can express exogenous aminoacyl-tRNA synthase mutants and exogenous orthogonal tRNA, so that the catalysis effect is exerted, and the alkenyl tyrosyl-tRNA product is obtained. The catalytic reaction is a bioorthogonal reaction, and compounds produced in host cells comprise natural amino acids and do not participate in the catalytic reaction; the products of the alkenyl tyrosine, exogenous orthogonal tyrosine tRNA and alkenyl tyrosyl tRNA required by the catalytic reaction do not affect the self-biochemical reaction and the vital activity of the host cell.
The advantageous effects of the present application will be explained in further detail below with reference to specific examples.
Example 1 construction of a screening protocol based on Red fluorescent protein and Chloramphenicol resistance
A screening scheme based on resistance of red fluorescent protein and chloramphenicol is constructed, and particularly, a coding gene of red fluorescent protein mcerry is adopted to replace a document to screen a green fluorescent protein gene GFPuv in a plasmid. Carrying out whole-gene synthesis by using a Jinzhi to obtain a codon-optimized mcherry sequence, introducing an NcoI enzyme cutting site at the 5 'end of the sequence, introducing an XhoI enzyme cutting site at the 3' end of the sequence, and constructing the sequence in a pET-28a vector, wherein the sequence is shown as SEQ ID NO:3, respectively.
SEQ ID NO：3：ccatgggcatgggcgttagcaaaggcgaagaagataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggcagcgtgaacggccatgagtttgaaatcgagggcgaaggcgaaggtcgcccatacgaaggcacccagaccgccaaactgaaagtgacgaaaggcggtccgctgccattcgcgtgggatattctgagcccacagttcatgtacggcagcaaagcctacgtgaagcatccggccgatatcccggattatctgaagctgagcttcccagagggcttcaagtgggagcgcgtgatgaactttgaagatggcggtgtggttaccgttacgcaagatagcagtctgcaagatggcgagttcatctacaaggttaagctccgcggcaccaacttcccgagcgatggtccggtgatgcagaaaaagacgatgggctgggaagcgagcagcgaacgcatgtatccagaagatggcgcgctgaaaggcgagatcaaacagcgtctgaagctgaaagacggcggccattacgatgcggaggtgaagaccacctacaaggcgaaaaagccggttcagctgccgggcgcgtacaacgtgaacatcaagctggacatcaccagccacaacgaagactacaccatcgtggagcagtacgaacgcgcggaaggtcgccatagtaccggcggcatggacgaactgtataaataatgactcgag。
BL21 (DE 3) competent cells were transformed with the expression plasmid, plated on LB + 50. Mu.g/mL kanamycin in solid LB medium, cultured overnight to obtain a monoclonal, inoculated in 5mL LB medium at 37 ℃ and 200rpm, cultured with shaking for 2-3 hours, and then OD 600 When it is 0.6-0.8After addition of a final concentration of 0.5mM IPTG and induction at 30 ℃ for 20h, the culture broth was found to turn purple-red, indicating that a red fluorescent protein was available. The sequences of the screening plasmids based on green fluorescence and antibiotics except for the coding sequence for green fluorescent protein were amplified by PCR using RFP-HR-Bone-F (SEQ ID NO:4 gagtcatgtatttcgcgggatgagcaggcaacgcaattatgaattagg) and RFP-HR-Bone-R (SEQ ID NO:5: (SEQ ID NO:6: (SEQ ID NO:7: cggtgatgacggtgaaaacaccaccgccgcgcgcttaatgc) as a primer, a mCherry coding frame sequence including a T7 promoter and a T7 terminator part is amplified by PCR (polymerase chain reaction), then a plasmid is constructed by homologous recombination, namely the replacement of fluorescent protein is completed, the constructed screening plasmid is transformed into DH10B competence, and a DH10B strain containing the screening plasmid is obtained by sequencing and is named as DH10B-REP (screening plasmid), so that the electrical transformation competence is prepared for later use.
EXAMPLE 2 construction of pET-Gln constitutive expression plasmid
In order to realize constitutive expression of aminoacyl tRNA synthase, a promoter part and a terminator part on pET-28a are replaced by a Gln promoter and a GlnTT terminator, the promoter and the terminator are synthesized into a DNA sequence by Jinwei, a BglII enzyme cutting site is introduced at the 5' end of the promoter, ndeI and EcoRI sites are introduced at the 3' end, an XhoI site is introduced at the 3' end of the GlnTT terminator, the synthesized sequence is connected with a pET28a vector through BglII and XhoI enzyme cutting, and pET-Gln is obtained through sequencing and is reserved.
The synthetic promoter and terminator sequences are shown in SEQ ID NO: shown in fig. 8.
SEQ ID NO：8：agatctgagctcccggtcatcaatcatccccataatccttgttagatgatcaattttaaaaaactaacagttcagcctgtcccgcttataagatccgttatacgtttacgctttgaggaatcccatcatatggaattcctgcagtttcaaacgctaaattgcctgatgcgctacgcttatcaggcctacatgatctctgatatattgagtacgtcttttgtaggccggataatcgttcactcgcatccggcagaaacagcaacatccaaaacgccgcgttcagcggcgtttatgcttttcttcgcgaattaattccgcttcgcaacatgtgagcaccggtttattgactaccggaagcagtgtgaccgtgtgctttaaatgcctgaggccagtttgctcaggctctccccgtggaggtaataattgacgatatgatcactcgag。
EXAMPLE 3 construction of pNFRS synthase mutant library
The amino acid sequence of pNFRS is shown in SEQ ID NO:1 is shown.
SEQ ID NO：1：
MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSSFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNPLNYEGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL。
The nucleotide sequence of the coding pNFRS is shown as SEQ ID NO:2, respectively.
SEQ ID NO 2：atggatgaatttgaaatgattaaacgcaacaccagcgaaattattagcgaagaagaactgcgcgaagtgctgaagaaggacgagaagtcagctctgattggctttgaaccgagcggcaagatacacctgggccattatttacagattaagaagatgatcgatttacagaacgcgggctttgatattattattctgctggcggatctgcatgcgtatctgaaccagaaaggcgaactggatgaaattcgcaagatcggagattataacaagaaggtatttgaggcgatgggcctgaaagcgaaatatgtgtatggcagcagctttcagctggataaagattataccctgaacgtgtatcgcctggcgctgaagacgacactgaaacgcgcgcgccgcagcatggaactgattgcgcgcgaagatgagaatcccaaagtggcggaagtgatttatccgattatgcaggtgaacccgctgaactatgaaggcgtggatgtggcggtgggcggcatggaacagcgcaagatacacatgctggcgcgcgaactgctgccgaagaaggtagtttgcattcataacccggtgctgaccggcctggatggcgaaggcaagatgtccagcagcaaaggcaactttattgcggtggatgatagcccggaagaaattcgcgcgaagatcaagaaagcgtactgcccggcgggcgtggtggaaggcaacccgattatggaaattgcgaaatatttcttagagtatccgctgaccattaaacgcccggagaagttcggtggcgatctgaccgtgaacagctatgaagaactggaaagcctgtttaagaataaggaactgcatccgatggatctgaagaatgctgtggcggaagaactgattaagatactcgaaccgattcgcaaacgcctgtaa。
Finding the distance between pNFRS and the para-hydroxyl of tyrosine through molecular docking
Multiple saturation mutations are carried out at internal amino acids (positions 31, 32, 65, 67, 69, 107, 108, 109, 110, 158, 159, 160, 161, 162). The primers used were:
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。
degenerate bases are present in the primers herein, wherein n represents any one of a, t, g, c, k represents g or t, m represents a or c.
Respectively amplifying three sections of 31-69, 65-110 and 107-162 by using the primers, and obtaining an NNK-containing fragment of pNFRS (31-162 aa) by over-lap PCR, wherein the vector part is expressed by SEQ ID NO:15: tgacttcgtcctctctagcacttcg (31-bone-R) and SEQ ID NO:16: ggatgtggcggtggtgggcggcgcgcgcatg (162-bone-F) is used as a primer and is obtained by PCR amplification. The described sections were placed here where the NNK fragment and vector sections were ligated by Circular Polymerase Extension Cloning (CPEC) as follows: respectively using 31-32NNK and 65-69NNK as primers to amplify a first fragment by PCR, using 65-69NNK and 107-110MNN as primers to amplify a second fragment by PCR, using 107-110NNK and 158-162MNN as primers to amplify a third fragment by PCR, then using 31-32NNK and 158-162MNN as primers to amplify a fused NNK fragment by overlap PCR, and finally connecting the fused fragment with a plasmid framework by a CPEC method. The recombinant vector is transformed into DH10B electric transformation competent cells by electric shock, kanamycin is added for overnight culture, a plasmid extraction kit is adopted to extract mutant mixed plasmids, and the mixed plasmids are stored at the temperature of-20 ℃ for standby.
Example 4 selection of mutant templates
To improve the specific introduction of unnatural amino acids and to improve the efficiency of the screening, reducing or eliminating the introduction of natural amino acids by aminoacyl-tRNA synthases will aid in the screening process. The wild type aminoacyl-tRNA synthase MjTyrRS from Methanodococcus jannaschii is initially adopted for screening, the specificity of the MjTyrRS is used for identifying the tyrosine Tyr which is a natural substrate, so the success rate during screening is not high, and the specificity of the mutation obtained by screening is poor, so the MjTyrRS mutant with low background is supposed to be beneficial to obtaining a novel high-efficiency specific aminoacyl-tRNA synthase mutant, and the discovery is made by combining literature research to find that the MjTyrRS mutant pNFRS can introduce p-nitro-L-phenylalanine pNF, p-iodo-L-phenylalanine pIF and tyrosine, but the specificity is poor. Therefore, a codon-optimized pNFRS sequence is synthesized, and based on the above embodiment, structural simulation and docking analysis are combined, N160H (nucleotide 478-480, prokaryotic nucleotide aac, mutated to cat-encoded His) and S207A (nucleotide 619-621, nucleotide agc, mutated to gcg-encoded alanine) are obtained through further mutation, amino acid introduction comparison is carried out, and a more appropriate template is found for mutation research.
pNFRS, pNFRS-160H and pNFRS-S207A coding sequences were ligated to pET-Gln, respectively, and screening plasmid REP based on red fluorescent protein was co-transformed into DH10B, which was cultured in LB medium with and without the addition of corresponding amino acids, and the results are shown in Table 1.
TABLE 1
Therefore, the optimal initial template is pNFRS-160H, and in order to verify the feasibility of the method, pNFRS and pNFRS-160H are respectively selected as templates to construct a multi-point mutation library for screening.
EXAMPLE 5 construction of pNFRS-160H synthase mutant library
From the results of the experiments in example 4, it was found that after the 160H mutation, specificity was enhanced by the introduction of the unnatural amino acid, thus creating a library of mutant synthases, which retained the 160H site, and other primers were used as in example 3, except for the 158-162MNN primer changes, such as SEQ ID NO: shown at 17.
SEQ ID NO：17：catgccgcccaccgccacatccacgccmnnmnnatgmnnmnngttcacctgcataatcggataaatcacttccgc。
Similarly, 3 short fragments were amplified by PCR, and the NNK-containing fragment of pNFRS-160H (31-162 aa) obtained by over-lap PCR was ligated to the vector CPEC, and then DH10B was electroporated into competent cells, cultured overnight with kanamycin, and the plasmids were extracted and stored at-20 ℃ for future use.
Example 6 screening of mutant libraries for pNFRS and pNFRS-160H
The synthase mutants obtained in examples 3 and 5, DH10B-REP were electrically transformed, recovered at 37 ℃ for 1 hour, plated on LB solid screening plate (hereinafter referred to as plate B) containing 10. Mu.g/mL tetracycline, 50. Mu.g/mL kanamycin, 50. Mu.g/mL chloramphenicol, 0.1% arabinose, 1mM OAY, and cultured, 48 hours to 72 hours until red colonies grew out, red colonies were simultaneously cultured on plate B and plate C (1 mM OAY on plate B was removed), colonies which did not grow on plate C, grown on plate B and had red fluorescence were streaked on plate B, red colonies were grown, and further screened for 2 to 3 rounds on plate B and plate C, and as a result, 1#,3#,8#,10#,12#,14# and 18# synthase mutants were screened from the pNFRS mutant library, 6#,13#,25#,30# and 35# synthase mutants were screened, and further, sequencing of the mutants was performed as shown in Table 2.
TABLE 2
Shake flask culture and comparison of the red clones-OAY is the group without unnatural amino acids, which corresponds to the addition of 1mM OAY, while determining OD 600 The values of (b) were used to calculate the fluorescence ratio per biomass, and the results are shown in Table 3.
TABLE 3
Example 7 construction of pNFRS and pNFRS-160H mutants and tRNA Co-expression plasmids
The coding sequence of TyrT, including promoter and terminator, is amplified from screening plasmid REP using tRNA-XhoI-F (SEQ ID NO:18 gggctcgagcacccaaaaaaaatactctcaac) and tRNA-HR-XhoI-R (SEQ ID NO: 19.
Example 8 evaluation of allyl-L-tyrosine incorporation
The codon-optimized green fluorescent protein sfGFP gene synthesized by Jinzhi is constructed at NcoI and XhoI sites of pACYCdue 1, and the sequence of the sfGFP gene is shown as SEQ ID NO: shown at 20.
SEQ ID NO：20：atgagcaaaggtgaagaactgtttaccggcgttgtgccgattctggtggaactggatggcgatgtgaacggtcacaaattcagcgtgcgtggtgaaggtgaaggcgatgccacgattggcaaactgacgctgaaatttatctgcaccaccggcaaactgccggtgccgtggccgacgctggtgaccaccctgacctatggcgttcagtgttttagtcgctatccggatcacatgaaacgtcacgatttctttaaatctgcaatgccggaaggctatgtgcaggaacgtacgattagctttaaagatgatggcaaatataaaacgcgcgccgttgtgaaatttgaaggcgataccctggtgaaccgcattgaactgaaaggcacggattttaaagaagatggcaatatcctgggccataaactggaatacaactttaatagccataatgtttatattacggcggataaacagaaaaatggcatcaaagcgaattttaccgttcgccataacgttgaagatggcagtgtgcagctggcagatcattatcagcagaataccccgattggtgatggtccggtgctgctgccggataatcattatctgagcacgcagaccgttctgtctaaagatccgaacgaaaaaggcacgcgggaccacatggttctgcacgaatatgtgaatgcggcaggtattacgtggagccatccgcagttcgaaaaa。
To achieve the introduction of an unnatural amino acid at a specific site in sfGFP, the 3-linked codon encoding I39 was mutated from att to tag, and the DNA sequencing yielded the pACYCdue-sfGFP (I39) plasmid.
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 and pACYCdiykuei-pNFRS-160H (35 #) -tRNA, pAT-Gln-pNFRS-160H (35 #) -tRNA and sCYfTfTun-pNFRS (39 #) -tG (37 #), are cultured in a single-LB) containing clone, LB (37 ℃ and cDNA) and cDNA (39) are cultured in a single-LB) as a single-strain. Selecting single clone, inoculating on 30mL LB (kan + Cm +1mM OAY), using LB (kan + Cm) without OAY as negative control, inducing with 1mM IPTG overnight at 30 deg.C, 485nm as excitation light, and 525nm emission light for measuring fluorescence intensity of green fluorescent protein, and determining OD 600 And calculating the fluorescence value of the unit bacterial concentration. And the literature-reported mutant synthase OAYRS-CK1 (32S, 107T,158T,159Y, 162A) (Santoro S W, wang L, herberich B, et al, an effective system for the evaluation of amino-tRNA synthetic specificity [ J]Nature Biotechnology,2002, 20 (10): 1044-1048.) and OAYRS-CKB (32Y, 107A,158C,159A, 162L) of this experimental design are compared and the results are shown in Table 4 and FIG. 1. And SEQ ID NO:1, and an enzyme that performs an N160H mutation based on the amino acid sequence of the pNFRS, cannot introduce OAY. From the results, the novel mutant carrying N160H has higher specificity of unnatural amino acid introduction.
TABLE 4
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: the aminoacyl-tRNA synthetase mutant can recognize alkenyl tyrosine with high specificity, catalyze the combination of the alkenyl tyrosine and corresponding tRNA to form alkenyl tyrosyl-tRNA, and can be applied to the fields of protein labeling, enzyme catalysis, protein and other molecule coupling and the like which are introduced and derived from the fixed point of unnatural amino acid in protein or polypeptide.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An aminoacyl-tRNA synthase mutant, comprising:
SEQ ID NO:1, wherein the mutation is selected from N160 mutation and any one or more of the following mutations:
the A31 mutation is A31S, A31G, A31C, A31T or A31L;
the L32 mutation is L32G, L32S, L32C, L32T, L32V, L32A or L32I;
the L65 mutation is L65V, L65T, L65C or L65I;
the A67 mutation is A67S;
l69 is mutated into 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;
the P158 mutation is P158A, P158L, P158S, P158D or P158M;
l159 is mutated to L159T, L159Q, L159E, L159V, L159D, L159I or L159A;
y161 is mutated to Y161M, Y161Q, Y161N, Y161S, Y161W or Y161F;
e162 mutations are E162V, E162M, E162L, E162T, E162I or E162D;
the N160 mutation is N160S, N160G, N160E, N160F, N160D, N160A, N160I, N160M, N160H or N160V.
2. The aminoacyl-tRNA synthase mutant according to claim 1, wherein the aminoacyl-tRNA synthase mutant is selected from the group consisting of SEQ ID NOs: 1, or a mutant which has any one of the following combinatorial mutations based on the amino acid sequence of pNFRS shown in 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。
3. a DNA molecule encoding the aminoacyl-tRNA synthase mutant according to claim 1 or 2.
4. A recombinant plasmid to which the DNA molecule of claim 3 is linked.
5. A host cell comprising the DNA molecule of claim 3 or the recombinant plasmid of claim 4.
6. The host cell of claim 5, wherein the host cell comprises a prokaryotic cell;
preferably, the prokaryotic cell comprises escherichia coli.
7. A method for producing an alkenyltyrosyl-tRNA, which comprises catalyzing the binding between an alkenyltyrosine (an unnatural amino acid) and a tRNA to produce the alkenyltyrosyl-tRNA, using the aminoacyl-tRNA synthase mutant according to claim 1 or 2.
8. The method of claim 7, wherein the alkenyl tyrosine comprises O-allyl-L-tyrosine.
9. The method according to claim 8, wherein the concentration of O-allyl-L-tyrosine is 1 to 5mM.
10. The method according to claim 7, wherein the binding of said alkenyltyrosine to said tRNA is catalyzed by said aminoacyl-tRNA synthase mutant contained in said host cell according to claim 5;
preferably, the 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.
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