Aminoacyl trna synthetase mutants and uses thereof

Abstract

The application discloses an aminoacyl tRNA synthetase mutant and application thereof. The aminoacyl tRNA synthetase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO:19, and the mutation at least includes the following mutation site: the 159th I is mutated into a hydroxyl-containing amino acid, a basic amino acid or a non-polar amino acid. The aminoacyl tRNA synthetase mutant has high activity and specificity, and can be used to realize the high-efficiency and site-specific introduction of non-natural amino acids in a protein.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and more specifically, to an aminoacyl-tRNA synthase mutant and its applications. Background Technology

[0002] Proteins in living organisms are composed of 20 naturally occurring amino acids arranged according to genetic information, performing a series of functions. However, the number of functional groups carried by these naturally occurring amino acids is limited, insufficient to meet the demands for protein structure and function in biological research and applications. In contrast, non-natural amino acids, carrying diverse functional groups, excel in protein modification. These non-natural amino acids contain ketone, aldehyde, azide, alkyne, amide, nitro, phosphate, and sulfonate groups, enabling them to undergo various modification reactions. This allows them to be applied in basic research, drug development, and bioengineering, for example, in detecting changes in protein structure, antibody-drug conjugates (ADCs), DNA-peptide conjugation, biosensors, peptide cyclization, fluorescent dye labeling, and molecular surface immobilization.

[0003] Synthetic techniques for encoding non-natural amino acids introduce these amino acids by adding additional components to the protein translation machine, including a set of orthogonal tRNAs, aminoacyl-tRNA synthetase (aaRS), and amber codons (TAG). The orthogonality of aaRS / tRNA requires that aaRS cannot recognize endogenous tRNAs or amino acids from the host, but can only aminoacylate their ligand tRNAs; similarly, tRNAs and target non-natural amino acids cannot be aminoacylated by endogenous aaRSs. A tyrosyl-tRNA synthetase / tRNA pair (MjTyrRS / TyrT) derived from the archaea *Methanocaldococcus jannaschii* has been modified for the site-directed introduction of over 60 non-natural amino acids. The alkyne group in alkyne-containing non-natural amino acids can undergo click chemistry reactions with other molecules containing azide groups, thus enabling its use in ADC drug development and protein labeling research. However, this requires multiple rounds of screening based on antibiotics and barnase nucleases, which is cumbersome and has not yielded highly efficient mutants. Summary of the Invention

[0004] The present invention aims to provide an aminoacyl-tRNA synthase mutant and its application to improve the efficiency of introducing non-natural amino acids.

[0005] To achieve the above objectives, according to one aspect of the present invention, an aminoacyl-tRNA synthase mutant is provided. This aminoacyl-tRNA synthase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO: 19, and the mutation includes at least the following mutation site: position 159I is mutated to a hydroxyl-containing amino acid, a basic amino acid, or a nonpolar amino acid.

[0006] Furthermore, the mutations include at least one of the following mutation sites: position 31, A, mutated to a nonpolar amino acid; position 32, Y, mutated to an aromatic or hydroxyl-containing amino acid; position 107, E, mutated to a negatively charged amino acid or a hydroxyl-containing amino acid; position 108, F, mutated to a basic amino acid; position 109, Q, mutated to a basic amino acid; position 110, L, mutated to methionine; position 158, D, mutated to an acidic amino acid, a hydroxyl-containing amino acid, or histidine; and position 160, H, mutated to a basic amino acid. The amino acid, hydroxyl-containing or aromatic amino acid, has a Y mutation at position 161 to an aromatic amino acid or a hydroxyl-containing amino acid; an L mutation at position 162 to a hydroxyl-containing amino acid or a nonpolar amino acid; preferably, the mutation includes at least one of the following combinations of mutation sites: L69I+E107D+F108R+Q109R+D158H+I159T+H160Q+Y161G+L162T; A31G+Y32S+E107S+D158T+I159 S+H160N+Y161S+L162T; Y32S+L69I+E107S+L110M+I159S; A31V+Y32T+L65C+F108R+Q109R+L110M +D158H+I159Y+H160S+Y161G+L162M; A31C+Y32T+E107S+D158S+I159Q+H160F+Y161S+L162T; Y32 S+L65I+E107D+F108K+Q109R+L110M+D158S+I159Q+H160F+Y161G+L162M;A31L+Y32F+L65I+F108R+L110M+D158T+I159L+H160S+Y161S+L162T;or A31V+E107S+Q109R+D158E+I159L+Y161W+L162T。

[0007] According to another aspect of the present invention, a DNA molecule is provided. This DNA molecule encodes any of the aforementioned aminoacyl-tRNA synthase mutants; preferably, the DNA molecule is formed by gene mutation based on the nucleotide sequence shown in SEQ ID NO: 7.

[0008] According to another aspect of the present invention, a recombinant plasmid is provided. This recombinant plasmid contains any of the aforementioned DNA molecules.

[0009] Furthermore, the recombinant plasmids are pET-22a(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b(+), pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+). +), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), p ET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32 , pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p -2. pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18 or pUC-19.

[0010] According to another aspect of the invention, a host cell is provided. This host cell contains any of the aforementioned recombinant plasmids.

[0011] Furthermore, the host cells include prokaryotic cells; preferably, the prokaryotic cells are Escherichia coli BL21-DE3 cells or Escherichia coli Rosetta-DE3 cells.

[0012] According to another aspect of the present invention, a method for introducing propynyl-L-tyrosine is provided. This method includes introducing propynyl-L-tyrosine using any of the above-mentioned aminoacyl-tRNA synthetase mutants or recombinant plasmids; preferably, it includes the following steps: constructing a recombinant plasmid containing any of the above-mentioned aminoacyl-tRNA synthetase mutants and the TyrT gene, and a recombinant plasmid containing a TAG codon at a specific site; co-transforming the two recombinant plasmids into host cells, culturing the host cells, and simultaneously adding propynyl-L-tyrosine; more preferably, the host cell is *Escherichia coli*, and during *E. coli* culture, the culture medium pH is 6.8–7.4, the culture temperature is 16–37°C, and the propynyl-L-tyrosine concentration is 1–10 mM.

[0013] The above-mentioned transaminyl-tRNA synthase mutant is based on the transaminyl-tRNA synthase shown in SEQ ID NO: 19. It is mutated by site-directed mutagenesis to change its amino acid sequence, thereby changing the protein structure and function. It has high activity and specificity. In addition, the application of this mutant can improve the efficiency of introducing non-natural amino acids. Attached Figure Description

[0014] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0015] Figure 1 The construction of a tyrosine tRNA synthase mutant library and the corresponding amino acid mutation sites are shown.

[0016] Figure 2 The fluorescence value per unit bacterial concentration is shown. Detailed Implementation

[0017] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0018] This invention improves upon the screening scheme based on green fluorescent protein and resistance genes described in the literature (An efficient system for the evolution of aminoacyl-tRNA synthetase specificity, Nature Biotechnology, volume 20, pages 1044–1048 (2002)). This improves upon the previous scheme, which relied on expensive flow cytometry, by replacing the previously inconspicuous green fluorescent protein with red fluorescent protein, achieving a visually identifiable screening scheme. Using this improved screening platform, to address the low efficiency of introducing propyne-L-tyrosine OpY* (structural formula below), a rationally designed multi-point saturation mutagenesis was conducted. After screening, mutants with more than a four-fold increase in introduction efficiency and improved specificity were finally obtained.

[0019]

[0020] According to a typical embodiment of the present invention, an aminoacyl-tRNA synthase mutant is provided. This aminoacyl-tRNA synthase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO: 19, and the mutation includes at least the following mutation site: position 159I is mutated to a hydroxyl-containing amino acid, a basic amino acid, or a nonpolar amino acid.

[0021] The above-mentioned transaminyl-tRNA synthase mutant of the present invention is based on the transaminyl-tRNA synthase shown in SEQ ID NO: 19, and is mutated by site-directed mutagenesis to change its amino acid sequence, thereby achieving changes in protein structure and function. It has high activity and specificity, and the application of this mutant can improve the efficiency of introducing non-natural amino acids.

[0022] To further improve enzyme activity and specificity, the mutations should include at least one of the following mutation sites: A at position 31 is mutated to a nonpolar amino acid; Y at position 32 is mutated to an aromatic or hydroxyl-containing amino acid; E at position 107 is mutated to a negatively charged amino acid or a hydroxyl-containing amino acid; F at position 108 is mutated to a basic amino acid; Q at position 109 is mutated to a basic amino acid; L at position 110 is mutated to methionine; D at position 158 is mutated to an acidic amino acid, a hydroxyl-containing amino acid, or histidine; H at position 160 is mutated to a basic amino acid, a hydroxyl-containing amino acid, or an aromatic amino acid; Y at position 161 is mutated to an aromatic amino acid or a hydroxyl-containing amino acid; and L at position 162 is mutated to a hydroxyl-containing amino acid or a nonpolar amino acid.

[0023] More preferably, the mutation includes at least one of the following combinations of mutation sites: L69I+E107D+F108R+Q109R+D158H+I159T+H160Q+Y161G+L162T; A31G+Y32S+E107S+D158T+I159S+H160N+Y161S+L162T; Y32S+L69I+E107S+L110M+I159S; A31V+Y32T+L65C+F108R+Q109R+L110M+D158H+I159Y+H160S+Y161G+L162 M; A31C+Y32T+E107S+D158S+I159Q+H160F+Y161S+L162T; Y32S+L65I+E107D+F108K+Q109R+L110M+D158S+I159Q+H160F+Y161G+L162M; A31L+Y32F+L65I+F108R+L110M+D158T+I159L+H160S+Y161S+L162T; or A31V+E107S+Q109R+D158E+I159L+Y161W+L162T.

[0024] According to a typical embodiment of the present invention, a DNA molecule is provided; preferably, the DNA molecule is formed by gene mutation based on the nucleotide sequence shown in SEQ ID NO: 7. This DNA molecule encodes the aforementioned aminoacyl-tRNA synthetase mutant. The aminoacyl-tRNA synthetase mutant encoded by this DNA molecule exhibits good specificity and activity.

[0025] The DNA molecules described above in this invention can also exist in the form of an "expression cassette." An "expression cassette" refers to a linear or circular nucleic acid molecule encompassing DNA and RNA sequences capable of directing the expression of a specific nucleotide sequence in an appropriate host cell. Generally, it includes a promoter effectively linked to the target nucleotide, optionally linked to a termination signal and / or other regulatory elements. The expression cassette may also include sequences required for the correct translation of the nucleotide sequence. The coding region typically encodes the target protein, but may also encode the target functional RNA, such as antisense RNA or untranslated RNA, in either the sense or antisense direction. Expression cassettes containing the target polynucleotide sequence can be chimeric, meaning that at least one of its components is heterologous to at least one of its other components. Expression cassettes can also be naturally occurring, but are obtained through efficient recombination for heterologous expression.

[0026] According to a typical embodiment of the present invention, a recombinant plasmid is provided. The recombinant plasmid contains any of the aforementioned DNA molecules. The DNA molecules in the recombinant plasmid are positioned at appropriate locations within the plasmid, enabling the DNA molecules to be correctly and smoothly replicated, transcribed, or expressed.

[0027] Although the present invention uses the qualifier "contains" when defining the aforementioned DNA molecule, this does not mean that other sequences unrelated to its function can be arbitrarily added to both ends of the DNA sequence. Those skilled in the art will know that, in order to meet the requirements of recombination operations, it is necessary to add suitable restriction endonuclease cleavage sites to both ends of the DNA sequence, or to add additional start codons, stop codons, etc. Therefore, using a closed-form description to define it will not truly cover these situations.

[0028] As used in this invention, the term "plasmid" includes any plasmid, granule, bacteriophage, or Agrobacterium binary nucleic acid molecule in double-stranded or single-stranded linear or circular form, preferably a recombinant expression plasmid, which can be a prokaryotic or eukaryotic expression plasmid, but is preferred to be a prokaryotic expression plasmid. In some embodiments, the recombinant plasmid is selected from pET-22a(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-1 2a(+), pET-14b(+), pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+) , pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET- 29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+ ), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE 2. pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX- 6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18 or pUC-19.

[0029] To facilitate subsequent screening, a red fluorescent protein reporter gene is operably linked to the recombinant plasmid; preferably, the red fluorescent protein reporter gene has the nucleotide sequence shown in SEQ ID NO: 1. Further, a chloramphenicol screening gene is operably linked to the recombinant plasmid; preferably, the recombinant plasmid is operably linked to a promoter and terminator sequence shown in SEQ ID NO: 6.

[0030] According to a typical embodiment of the present invention, a host cell is provided, the host cell containing any of the above-described recombinant plasmids. Host cells suitable for the present invention include, but are not limited to, prokaryotic cells. Preferably, the prokaryotic cell is *Escherichia coli* BL21-DE3 cells or *Escherichia coli* Rosetta-DE3 cells.

[0031] According to a typical embodiment of the present invention, a method for introducing propynyl-L-tyrosine is provided. The method includes introducing propynyl-L-tyrosine using an aminoacyl-tRNA synthetase mutant or the recombinant plasmid described above. Preferably, the method includes the following steps: constructing a recombinant plasmid containing any of the above-mentioned aminoacyl-tRNA synthetase mutants and the TyrT gene, and a recombinant plasmid containing a TAG codon at a specific site; co-transforming the two recombinant plasmids into host cells, culturing the host cells, and simultaneously adding propynyl-L-tyrosine; more preferably, the host cell is *Escherichia coli*, and during *E. coli* culture, the culture medium pH is 6.8–7.4, the culture temperature is 16–37°C, and the propynyl-L-tyrosine concentration is 1–10 mM.

[0032] More specifically, in a preferred embodiment of the present invention, the method for introducing propynyl-L-tyrosine includes: 1) constructing a plasmid containing the MjTyrRS mutant and TyrT of the present invention. In this embodiment, a constitutive promoter is used, but inducible promoters such as T7 or Sp6 phage-derived promoters can also be used; 2) expressing the target gene sfGFP in a plasmid (the fluorescent protein used for testing; this method can also be applied to any other target protein). The T7 inducible promoter is used, but it can also be replaced with other inducible promoters, including Sp6, xylose inducible promoters, arabinose inducible promoters, etc.; 3) co-transforming the two plasmids into E. coli. The host currently used is BL21(DE3), but it can also be extended to any other E. coli cells; 4) culturing E. coli, adding non-natural amino acids at a concentration of 1-10 mM during induction, and detecting fluorescence after culturing. Preferably, the plasmids contain the MjTyrRS mutant and TyrT, and the plasmid contains the target gene, which includes one or more TAG mutation sites to introduce the corresponding non-natural amino acid. The two plasmids must contain different replication origin sites to ensure their simultaneous stable existence. When culturing *E. coli*, the pH should be 6.8–7.4, and the culture temperature 16–37°C. The non-natural amino acid propynyl-L-tyrosine needs to be added. When dissolved in water, NaOH needs to be added to adjust the pH to approximately 10.0 for complete dissolution. Then, the solution is filtered to remove bacteria and added to the culture medium at a final concentration of 1–10 mM.

[0033] The beneficial effects of the present invention will be further illustrated below with reference to embodiments.

[0034] Example 1

[0035] Construct a screening scheme based on red fluorescent protein and chloramphenicol resistance

[0036] A screening scheme based on red fluorescent protein and chloramphenicol resistance was constructed. Specifically, the coding gene of the red fluorescent protein mcherry was used to replace the green fluorescent protein gene GFPuv in the plasmid for screening (An efficient system for the evolution of aminoacyl-tRNA synthetase specificity Nature Biotechnology volume 20, pages 1044–1048 (2002)). The whole genome of mcherry was synthesized by Genewiz and codon-optimized. An NcoI restriction site was introduced at the 5' end of the sequence and an XhoI restriction site was introduced at the 3' end of the sequence. The sequence was constructed in the pET-28a vector as follows (SEQ ID NO: 1):

[0037] .

[0038] The expression plasmid was transformed into BL21(DE3) competent cells, plated on LB agar plates containing 50 μg / mL kanamycin, and cultured overnight to obtain single clones. The single clones were then seeded into 5 ml of LB agar and cultured at 37°C with shaking at 200 rpm for 2-3 h. When the OD600 reached 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and the cells were induced at 30°C for 20 h. The culture medium turned purple-red, indicating that the red fluorescent protein was usable. Using RFP-HR-Bone-F (SEQ ID NO: 2, GAGTCGTATTAATTTCGCGGGATCGAGTGAGCGCAACGCAATTAATG) and RFP-HR-Bone-R (SEQ ID NO: 3, GCATTAAGCGCGGCGGGTGTGGTGTTTTCACCGTCATCACCG) as primers, the sequence other than the green fluorescent protein coding sequence of the green fluorescence and antibiotic screening plasmid was amplified by PCR (Nat Biotechnol. 2002 Oct; 20(10):1044-1048.). Using RFP-HR-F (SEQ ID NO: 4, CATTAATTGCGTTGCGCTCACTCGATCCCGCGAAATTAATACGACTC) and RFP-HR-R (SEQ ID NO: 4, CATTAATTGCGTTGCGCTCACTCGATCCCGCGAAATTAATACGACTC) as primers, the sequence of the green fluorescent protein-based screening plasmid was amplified by PCR. Using primers (NO: 5, CGGTGATGACGGTGAAAACACCACACCCGCCGCGCTTAATGC), the mCherry coding frame sequence, including the T7 promoter and T7 terminator, was amplified by PCR. Then, a plasmid was constructed through homologous recombination, thus completing the replacement of the fluorescent protein. The constructed selection plasmid was transformed into DH10B competent cells. After sequencing, DH10B strains containing the selection plasmid were obtained and named DH10B-REP (selection plasmid). These were then prepared as electrotransformation competent cells for later use.

[0039] Real-time Example 2

[0040] Construction of pET-Gln constitutive expression plasmid

[0041] To achieve constitutive expression of aminoacyl-tRNA synthase, the promoter and terminator portions of pET-28a were replaced with the Gln promoter and GlnTT terminator. The promoter and terminator were synthesized into a single DNA sequence by Genewiz. A BglII (AGATCT) restriction site was introduced at the 5' end of the promoter, and NdeI (CATATG) and EcoRI (GAATTC) sites were introduced at the 3' end. An XhoI (CTCGAG) site was introduced at the 3' end of the GlnTT terminator. The synthesized sequence was ligated into the pET28a vector by digestion with BglII and XhoI, and pET-Gln was obtained by sequencing for later use.

[0042] The synthesized promoter and terminator sequences are as follows (SEQ ID NO: 6):

[0043] AGATCT gagctcccggtcatcaatcatccccataatccttgttagatgatcaattttaaaaaactaacagttcagcctgtcccgcttataagatccgttatacgtttacgctttgaggaatcccat CATATGGAATTC ctgcagtttcaaacgctaaattgcctgatgcgctacgcttatcaggcctacatgatctctgatatattgagtacgtcttttgtaggccggataatcgttcactcgcatccggcagaaacagcaacatccaaaacgccgcgttcagcggcg tttatgcttttcttcgcgaattaattccgcttcgcaacatgtgagcaccggtttattgactaccggaagcagtgtgaccgtgtgctttaaatgcctgaggccagtttgctcaggctctccccgtggaggtaataattgacgatatgatca CTCGAG .

[0044] Example 3

[0045] Constructing a wild-type expression plasmid for tyrosine-tRNA synthase

[0046] The MjTyrRS sequence, optimized by Genewiz's synthetic codons, is as follows (SEQ ID NO: 7):

[0047] ATGGATGAATTTGAAATGATTAAACGCAACACCAGCGAAATTATTAGCGAAGAAGAACTGCGCGAAGTGCTGAAGAAGGACGAGAAGTCAGCTTATATTGGCTTTGAACCGAGCGGCAAGATACACCTGGGCCATTATTTACAGATTAAGAAGATGATCGATTTACAGAACGCGGGCTTTGATATTATTATTCTGCTGGCGGATCTGCATGCGTATCTGAACCAGAAAGGCGAACTGGATGAAATTCGCAAGATCGGAGATTATAACAAGAAGGTATTTGAGGCGATGGGCCTGAAAGCGAAATATGTGTATGGCAGCGAGTTTCAGCTGGATAAAGATTATACCCTGAACGTGTATCGCCTGGCGCTGAAGACGACACTGAAACGCGCGCGCCGCAGCATGGAACTGATTGCGCGCGAAGATGAGAATCCCAAAGTGGCGGAAGTGATTTATCCGATTATGCAGGTGAACGATATTCACTATCTGGGCGTGGATGTGGCGGTGGGCGGCATGGAACAGCGCAAGATACACATGCTGGCGCGCGAACTGCTGCCGAAGAAGGTAGTTTGCATTCATAACCCGGTGCTGACCGGCCTGGATGGCGAAGGCAAGATGTCCAGCAGCAAAGGCAACTTTATTGCGGTGGATGATAGCCCGGAAGAAATTCGCGCGAAGATCAAGAAAGCGTACTGCCCGGCGGGCGTGGTGGAAGGCAACCCGATTATGGAAATTGCGAAATATTTCTTAGAGTATCCGCTGACCATTAAACGCCCGGAGAAGTTCGGTGGCGATCTGACCGTGAACAGCTATGAAGAACTGGAAAGCCTGTTTAAGAATAAGGAACTGCATCCGATGGATCTGAAGAATGCTGTGGCGGAAGAACTGATTAAGATACTCGAACCGATTCGCAAACGCCTGTAA

[0048] After digestion with NdeI and EcoRI, it was constructed into the pET-Gln vector to obtain the pET-Gln-MjTyrRS synthase expression plasmid.

[0049] The protein sequence encoded by MjTyrRS is as follows (SEQ ID NO: 19):

[0050] MDEFEMIKRNTSEIISEEELREVLKKDEKSAYIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPI MQVNDIHYLGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL.

[0051] Example 4

[0052] Construction of a tyrosine-tRNA synthase mutant library

[0053] Molecular docking was used to locate the para-hydroxyl group of tyrosine on MjTyrRS. Multiple-point saturation mutagenesis was performed on the amino acids within the range of 31, 32, 65, 67, 69, 107, 108, 109, 110, 158, 159, 160, 161, 162. Primers used:

[0054] 31-32NNK (SEQ ID NO: 8): GCTGAAGAGGACGAGAAGTCANNKNNKATTGGCTTTGAACCGAGCGGCAAGATAC;

[0055] 65-69-MNN (SEQ ID NO: 9):TCTGGTTCAGATACGCATGMNNNATCMNNCAGMNNAATAATAATATCAAAGCCCGCGTTCTG;

[0056] 65-69NNK (SEQ ID NO: 10): GGGCTTTGATATTATTNNKCTGNNKGATNNKCATGCGTATCTGAACCAGAAAGGCGAACTG;

[0057] 107-110MNN (SEQ ID NO: 11):CACGTTCAGGGTATAATCTTTATCMNNMNNMNNMNNGCTGCCATACACATATTTCGCTTTCAGGCCCATC;

[0058] 107-110NNK (SEQ ID NO: 12): GCGAAATATGTGTATGGCAGCNNKNNKNNKNNKGATAAAGATTATACCCTGAACGTGTATCGCCTG;

[0059] 158-162MNN (SEQ ID NO: 13): CATGCCGCCCACCGCCACATCCACGCCMNNMNNMNNMNNMNNGTTCACCTGCATAATCGGATAAATCACTTCCGC.

[0060] like Figure 1 As shown, the three fragments 31-69, 65-110, and 107-162 were amplified using the primers above, and then the NNK-containing fragment of MjTyrRS (31-162aa) was obtained by over-lap PCR. The vector portion was obtained by PCR amplification using primers 31-bone-R (SEQ ID NO: 14): TGACTTCTCGTCCTTCTTCAGCACTTCG and 162-bone-F (SEQ ID NO: 15): GGATGTGGCGGTGGGCGGCATG. The NNK fragment and the vector portion pET-Gln-MjTyrRS were ligated using the circular polymerase extension cloning (CPEC) method. The recombinant vector was electroporated into DH10B electroporated competent cells, and kanamycin was added for overnight culture. The mutant mixed plasmid was extracted using a plasmid extraction kit and stored at -20℃ for later use.

[0061] Example 5

[0062] Screening for tyrosyl-tRNA synthase mutants

[0063] The tyrosyl-tRNA synthase mutant obtained in Example 4 was electroporated into DH10B-REP, and after recovery at 37°C for 1 h, it was plated on LB solid selection 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 OpY. The plates were cultured for 48-72 h until red clones appeared. The red clones were simultaneously cultured on B plates and C plates (with the 1 mM OpY removed from the B plate). Clones that did not grow on the C plate but grew on the B plate and exhibited red fluorescence were streaked on the B plate. After the red clones grew, further selection was performed on B and C plates for 2-3 rounds to finally obtain the tyrosyl-tRNA synthase mutant that could efficiently introduce OpY. The mutant was sequenced to obtain the coding sequence and mutation site. The information is shown in Table 1.

[0064] Table 1

[0065]

[0066]

[0067] Example 6

[0068] Construction of fluorescent protein recombinant plasmid

[0069] The green fluorescent protein sfGFP gene (for testing) was synthesized and codon optimized by Genewiz. It was constructed at the NcoI and XhoI sites of pACYCduet1. The sfGFP gene sequence is as follows (SEQ ID NO: 16):

[0070] To introduce non-natural amino acids into a specific site in sfGFP, the triplet codon encoding I39 was mutated from ATT to TAG, and DNA sequencing yielded the pACYCduet-sfGFP(I39) plasmid.

[0071] Example 7

[0072] Construct plasmids containing the MjTyrRS mutant and TyrT

[0073] The coding sequence of TyrT, including the promoter and terminator, was amplified from the screening plasmid REP using tRNA-XhoI-F (SEQ ID NO: 17, gggCTCGAGCCCATCAAAAAAATATTCTCAAC) and tRNA-HR-XhoI-R (SEQ ID NO: 18, gggCTCGAGtaaaaaaaatccttagctttcg). The product was digested with XhoI and ligated with pET-Gln-MjTyrRS (obtained by sequencing). DNA sequencing was then performed to obtain the pET-Gln-MyTyrRS(mut)-tRNA plasmid.

[0074] Example 8

[0075] Evaluation of propynyl-L-tyrosine introduction

[0076] pET-Gln-MyTyrRS(2#)-tRNA, pET-Gln-MyTyrRS(5#)-tRNA, pET-Gln-MyTyrRS(16#)-tRNA, pET-Gln-MyTyrRS(32#)-tRNA, pET-Gln-MyTyrRS(55#)-tRNA, pET-Gln-MyTyrRS(62#)-tRNA, pET-Gln-MyTyrRS(75#)-tRNA, and pET-Gln-MyTyrRS(88#)-tRNA were co-transformed with pACYCduet-sfGFP(I39TAG) into BL21(DE3) competent cells. The cells were then plated on LB culture plates containing kanamycin and chloramphenicol and cultured at 37°C until single colonies grew. Single colonies were selected and inoculated into 30 ml of LB (kan+Cm+1mM OpY), with LB (kan+Cm) without OpY as a negative control. After induction overnight at 30℃ with 1 mM IPTG, the samples and positive control cells turned bright green, indicating good expression. The negative control showed no visible fluorescence. The fluorescence intensity of the green fluorescent protein was measured using 485 nm excitation light and 525 nm emission light, and the OD600 was also measured. The fluorescence value per unit bacterial concentration was calculated. Comparison with reported mutant synthases OpYRS (32A, 107P, 158A, 162A) in the literature yielded the following results: Figure 2 .

[0077] Compared with the literature (In vivo incorporation of an alkyne into proteins in Escherichia coli Bioorganic & Medicinal Chemistry Letters 15(2005)1521–1524) OpYRS, the mutants OpYRS-2#, OpYRS-5#, OpYRS-16#, OpYRS-32#, OpYRS-55#, OpYRS-62#, OpYRS-75#, and OpYRS-88# obtained in this invention showed increased activities of 4.97, 4.57, 4.10, 5.0, 3.85, 5.83, 4.07, and 5.42 times, respectively, and their specificity was also significantly improved. The fluorescence values ​​were significantly improved (fluorescence value with OpY added / OD600: fluorescence value without OpY added / OD600): OpYRS-2# = 20.46, OpYRS-5# = 16.9, OpYRS-16# = 15.8, OpYRS-32# = 9.1, OpYRS-55# = 13.2, OpYRS-62# = 13.5, OpYRS-75# = 16.2, OpYRS-88# = 10.8, while the control was only 5.72.

[0078] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects:

[0079] This invention optimizes the screening scheme based on red fluorescence and antibiotic resistance, enabling visually identifiable clone screening. Based on the results of structure and molecular docking, a tyrosyl-tRNA synthase mutant library covering 14 sites was constructed. Furthermore, based on the improved screening scheme, eight synthase mutants that can efficiently introduce OpY were obtained. Compared with the literature, the new synthase mutants obtained have significantly improved in both activity and specificity.

[0080] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An aminoacyl-tRNA synthase mutant, characterized in that, The aminoacyl-tRNA synthase mutant was obtained by mutating the amino acid sequence shown in SEQ ID NO: 19, and the mutation site was: L69I+E107D+F108R+Q109R+D158H+I159T+H160Q+Y161G+L162T. A31G+Y32S+E107S+D158T+I159S+H160N+Y161S+L162T; Y32S+L69I+E107S+L110M+I159S; A31V+Y32T+L65C+F1 08R+Q109R+L110M+D158H+I159Y+H160S+Y161G+L162M; A31C+Y32T+E107S+D158S+I159Q+H160F+Y161S+L162T; Y32S+L65I+E107D+F108K+Q109R+L110M+D158S+I159Q+H160F+Y161G+L162M; A31L+Y32F+L65I+F108R+L110M+D158T+I159L+H160S+Y161S+L162T; or A31V+E107S+Q109R+D158E+I159L+Y161W+L162T.

2. A DNA molecule, characterized in that, The DNA molecule encodes the aminoacyl-tRNA synthase mutant of claim 1.

3. The DNA molecule according to claim 2, characterized in that, The DNA molecule is formed by gene mutation based on the nucleotide sequence shown in SEQ ID NO:

7.

4. A recombinant plasmid, characterized in that, The recombinant plasmid contains the DNA molecule as described in claim 2 or 3.

5. The recombinant plasmid according to claim 4, characterized in that, The recombinant plasmids are pET-22a(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b(+), pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), and pET-35b(+). ), pET-38b (+), pET-39b (+), pET-40b (+), pET-41a (+), pET-41b (+), pET-42a (+), p ET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32 , pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p -2. pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18 or pUC-19.

6. A host cell, characterized in that, The host cell contains the recombinant plasmid as described in claim 4 or 5.

7. The host cell according to claim 6, characterized in that, The host cells include prokaryotic cells.

8. The host cell according to claim 7, characterized in that, The prokaryotic cells are either Escherichia coli BL21-DE3 cells or Escherichia coli Rosetta-DE3 cells.

9. A method for introducing propynyl-L-tyrosine, characterized in that, This includes introducing propynyl-L-tyrosine using the aminoacyl-tRNA synthetase mutant as described in claim 1 or the recombinant plasmid as described in any one of claims 4 or 5.

10. The method for introducing propynyl-L-tyrosine according to claim 9, characterized in that, Includes the following steps: Construct the recombinant plasmid as described in claim 4 or 5 and the recombinant plasmid containing the TyrT gene; The two recombinant plasmids were co-transformed into host cells, the host cells were cultured, and propynyl-L-tyrosine was added simultaneously.

11. The method for introducing propynyl-L-tyrosine according to claim 10, characterized in that, The host cell is Escherichia coli. When culturing E. coli, the culture medium pH is 6.8~7.4, the culture temperature is 16~37℃, and the concentration of propynyl-L-tyrosine is 1~10 mM.

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