An aminoacyl-tRNA synthetase mutant and use thereof
By using gene codon expansion technology and the design of aminoacyl-tRNA synthetase mutants, the problem of 2-methylalanine synthesis in vivo was solved, enabling the specific introduction of non-natural amino acids into Escherichia coli and the improvement of protein function.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHUHAI SHENGTIDE INVESTMENT PARTNERSHIP (LLP)
- Filing Date
- 2026-03-17
- Publication Date
- 2026-06-02
AI Technical Summary
Existing technologies cannot efficiently and economically utilize 2-methylalanine directly in organisms to participate in peptide/protein synthesis. Chemical methods are costly, have low yields, and pollute the environment.
Using gene codon expansion technology, the 2-methylalanine (Aib) translation system MjAibRS/tRNACUA was screened out, and the wild-type tyrosine-tRNA synthetase of Methanococcus japonicus was directed to evolve to prepare aminoacyl-tRNA synthetase mutants, thereby achieving the specific introduction of non-natural amino acids into Escherichia coli.
We successfully expressed the green fluorescent protein of 2-methylalanine and the intermediate smegglutinin in Escherichia coli, which improved the half-life of amino acids and protein function, and reduced the synthesis cost.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering, specifically to an aminoacyl-tRNA synthetase mutant and its applications. Background Technology
[0002] Throughout evolution, organisms have enriched the structure and function of proteins by altering the sequence of amino acids. Since organisms themselves encode only 20 natural amino acids, non-natural amino acids cannot be inserted into protein polypeptide chains via genetic coding. By designing and modifying the translation system in the central dogma, codon expansion technology can specifically introduce non-natural amino acids into designated sites on target proteins within cells. Utilizing the unique functional groups within these non-natural amino acids, the target protein acquires new physicochemical properties, ultimately achieving the goal of protein functional innovation.
[0003] Aminoacyl-tRNA synthetases strictly adhere to the genetic code table during translation, specifically recognizing corresponding amino acids, thus ensuring the orthogonality of aminoacyl-tRNA synthetases to their substrates. Therefore, developing tool enzymes that specifically recognize non-natural amino acids requires the design, modification, and directed evolution of aminoacyl-tRNA synthetases. Using this strategy, over 200 non-natural amino acids have been specifically introduced into biological proteins.
[0004] 2-Methylalanine, also known as 2-aminoisobutyric acid (Aib), is a non-natural amino acid that can be used to modify polypeptides / proteins to obtain new biological functions, such as eliminating protease cleavage sites, prolonging half-life, reducing antigenicity, and stabilizing α-helix structures. Smegglutide, tasglutide, abalotide, and transmembrane peptides have all achieved better drug properties through Aib modification. Taking smegglutide as an example, dipeptidyl peptidase IV (DPP-IV) in the human body can cleave between the Ala8 and Glu9 amino acids, resulting in a shorter plasma half-life for GLP-1. Replacing Ala with non-natural Aib significantly increases the half-life, allowing the dosing frequency to change from once daily to once weekly.
[0005] There are currently no reports on 2-methylalanine directly participating in polypeptide / protein synthesis in organisms. It is introduced through chemical methods. In actual production applications, it takes a long time to explore the synthetic route, which is costly, has low yield, and pollutes the environment. Summary of the Invention
[0006] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide an aminoacyl-tRNA synthetase mutant and its application.
[0007] The technical solution adopted in this invention is:
[0008] In a first aspect, the present invention provides an aminoacyl-tRNA synthetase mutant, wherein the aminoacyl-tRNA synthetase mutant is any mutation of the amino acid sequence of the wild-type tyrosine-tRNA synthetase from *Methanococcus japonicus* at any of the following sites:
[0009] 1) Form 1: Replace position 32 with glycine, position 53 with alanine, position 75 with glutamic acid, position 158 with threonine, and position 204 with arginine;
[0010] 2) Form 2: 32 is replaced with glutamine, 53 with alanine, 75 with lysine, 158 with glutamic acid, and 204 with serine;
[0011] 3) Form 3: Replace position 32 with glycine, position 53 with aspartic acid, position 75 with arginine, position 158 with glutamic acid, and position 204 with serine;
[0012] 4) Form 4: 32 is replaced with glutamine, 53 with alanine, 75 with lysine, 158 with threonine, and 204 with arginine;
[0013] 5) Form 5: Replace position 32 with glycine, position 53 with aspartic acid, position 75 with glutamic acid, position 158 with threonine, and position 204 with serine;
[0014] 6) Form 6: Replace position 32 with glycine, position 53 with valine, position 75 with glutamic acid, position 158 with threonine, and position 204 with arginine.
[0015] Specifically, the amino acid sequence of the aminoacyl-tRNA synthetase mutant is shown in SEQ ID No. 37-42.
[0016] Specifically, the amino acid sequence of the wild-type tyrosine-tRNA synthetase from Methanococcus japonicus is shown in SEQ ID No. 2.
[0017] In a second aspect, the present invention provides a gene encoding the aminoacyl-tRNA synthetase mutant described in the first aspect.
[0018] Thirdly, the present invention provides a recombinant expression vector comprising the gene described in the second aspect.
[0019] Fourthly, the present invention provides a genetically engineered bacterium, wherein the genetically engineered bacterium comprises the recombinant expression vector described in the third aspect.
[0020] Fifthly, the present invention provides the use of the aminoacyl-tRNA synthetase mutant described in the first aspect in the preparation of recombinant proteins containing non-natural amino acids.
[0021] Specifically, the non-natural amino acid is 2-aminoisobutyric acid.
[0022] Specifically, the recombinant protein is selected from any one of smegglutinin, tasmoglutinin, abaloidin, and membrane-penetrating peptides.
[0023] The beneficial effects of this invention are:
[0024] To address the current inability to directly utilize 2-methylalanine in protein synthesis within organisms, a 2-methylalanine (Aib) translation system, MjAibRS / tRNACUA, was successfully screened using gene codon expansion technology. This translation system was then used to successfully express green fluorescent protein containing the non-natural amino acid 2-methylalanine and smegglutinin intermediates in Escherichia coli. Attached Figure Description
[0025] Figure 1 This is a vector map of the plasmid pRS-WT containing wild-type MjTyrRS in this invention.
[0026] Figure 2 This is a vector map containing plasmid pCM-RFP in this invention.
[0027] Figure 3 This is a vector map containing plasmid pCM-BAR in this invention.
[0028] Figure 4 The image shows the results of the Aib-dependent RFP fluorescence detection method in this invention.
[0029] Figure 5 This is a comparison of the relative fluorescence values of EGFP in E. coli with and without the addition of Aib according to the present invention.
[0030] Figure 6 This is a spectrum of SDS-PAGE detection results from Example 4 of the present invention. Detailed Implementation
[0031] This invention will be more readily understood through the following detailed description of some embodiments of the invention and the examples included therein. Before further describing the invention, it should be understood that the invention is not limited to the specific embodiments described, as these embodiments are necessarily diverse. It should also be understood that the terminology used in this specification is for illustrative purposes only and is not intended to be limiting, as the scope of the invention will be defined solely by the appended claims.
[0032] The tyrosine-tRNA synthetase / tyrosine-tRNA (MjTyrRS / tRNATyr) orthogonal pair from *Methanococcus janaschii* was initially introduced into *Escherichia coli* to encode proteins containing O-methyl-L-tyrosine. The reasons for choosing this orthogonal pair in *E. coli* include: ① The first base pair on the amino acid acceptor stem of MjTRNATyr is CG (GC in *E. coli*), effectively preventing recognition by endogenous TyrRS enzymes; ② MjTyrRS lacks an editing and proofreading mechanism for substrate amino acids, facilitating the introduction of non-natural amino acids. These characteristics greatly reduced the difficulty of modifying orthogonal tools, and the modified orthogonal pair has been widely used due to its good orthogonality and high activity in *E. coli*.
[0033] All reagents used in the following examples were analytical grade. Molecular cloning-related enzyme reagents were purchased from New England Biolabs. The *E. coli* strains were preserved in our laboratory. Gene synthesis, primer synthesis, and gene sequencing were performed by Suzhou Genewiz Biotechnology Co., Ltd. Unless otherwise specified in the examples, standard conditions or manufacturer-recommended conditions were followed. Reagents and instruments whose manufacturers are not specified are commercially available products. Molecular cloning experimental procedures not described in detail in the examples are based on *Molecular Cloning: A Laboratory Manual (4th Edition)* (edited by M.R. Green and J. Sambrook, translated by He Fuchu, Beijing: Science Press, 2017).
[0034] Example 1 (Construction of a mutant library)
[0035] This embodiment describes the construction of plasmid pRS for a mutant library containing aminoacyl-tRNA synthetase (MjAibRS). The plasmid pRS for the mutant library mainly consists of an asparagine tRNA synthetase promoter (GlnRS promoter), a GlnRS terminator, 32Tyr, 53Ile, 75Gln, 158Asp, 204Lys mutant MjTyrRS, a high-copy ColE1 replicon, and an ampicillin resistance gene.
[0036] (1) Constructing the plasmid pRS-WT containing MjTyrRS wild-type
[0037] The wild-type MjTyrRS gene sequence was obtained from NCBI Reference SEQuence: WP_010869888.1, as shown in SEQ ID No. 1, and its encoded amino acid sequence is shown in SEQ ID No. 2. The GlnRS promoter gene coding sequence is shown in SEQ ID No. 3, and the GlnRS terminator gene coding sequence is shown in SEQ ID No. 4. The high-copy ColE1 replicon and ampicillin resistance gene were obtained from the commercial pUC19 vector. pUC19 was ligated to the gene coding sequences SEQ ID No. 3, SEQ ID No. 1, and SEQ ID No. 4. NdeI and PstI restriction sites were inserted before and after SEQ ID No. 1 of the MjTyrRS coding gene, respectively, resulting in the vector map shown below. Figure 1 The plasmid pRS-WT containing wild-type MjTyrRS shown is sequenced in SEQ ID No. 5. This sequence was fully synthesized by Suzhou Genewiz Biotechnology Co., Ltd.
[0038] (2) Constructing plasmid pRS containing the MjAibRS mutant library
[0039] Starting with the wild-type plasmid pRS-WT from (1), the MjAibRS mutant library was constructed through site-directed mutagenesis and three rounds of polymerase chain reaction. The mutation sites included 32Tyr, 53Ile, 75Gln, 158Asp, and 204Lys. The plasmid containing this mutant library was named pRS. The primers required for the site-directed mutagenesis method are shown in Table 1.
[0040] The first round of mutations involved the 53Ile and 158Asp sites. Primers pRS-P4 and pRS-P6 contained degenerate codons at the 53Ile and 158Asp sites, respectively. Using pRS-WT as a template, PCR amplification was performed using three primer pairs: pRS-P1 / pRS-P4, pRS-P3 / pRS-P5, and pRS-P6 / pRS-P2. The amplification lengths were 174 bp, 312 bp, and 467 bp, respectively. The fragments were separated and recovered by agarose gel electrophoresis. The plasmid pRS-WT was digested with NdeI and PstI to obtain two fragments: the wild-type MjTyrRS (927 bp) and the pRS vector backbone NdeI-pRS-PstI (2286 bp). The NdeI-pRS-PstI fragment was separated and recovered by agarose gel electrophoresis. The recovered pRS-P1 / pRS-P4, pRS-P3 / pRS-P5, pRS-P6 / pRS-P2, and NdeI-pRS-PstI fragments were ligated using the Gibson ligation method to form a circular plasmid pRS-M1 library containing mutations at the 53Ile and 158Asp sites, completing the first round of mutations.
[0041] The second round of mutations involved the 75Gln and 204Lys sites. Primers pRS-P7 and pRS-P9 contained degenerate codons with mutations at the 75Gln and 204Lys sites, respectively. Using the pRS-M1 library as a template, PCR amplification lengths of 241 bp, 404 bp, and 309 bp were achieved using three primer pairs: pRS-P1 / pRS-P7, pRS-P8 / pRS-P9, and pRS-P10 / pRS-P2. The fragments were then separated and recovered by agarose gel electrophoresis. The recovered pRS-P1 / pRS-P7, pRS-P8 / pRS-P9, pRS-P10 / pRS-P2, and NdeI-pRS-PstI fragments were ligated using Gibson ligation to form the circular plasmid pRS-M2 library containing mutations at the 53Ile, 75Gln, 158Asp, and 204Lys sites, thus completing the second round of mutations.
[0042] The third round of mutations involved the 32Tyr site, with primer pRS-P12 containing a degenerate codon from the 32Tyr mutation. Using the pRS-M2 library as a template, PCR amplification was performed using two primer pairs, pRS-P1 / pRS-P12 and pRS-P11 / pRS-P2, amplifying lengths of 112 bp and 825 bp, respectively. The fragments were then separated and recovered by agarose gel electrophoresis. The recovered pRS-P1 / pRS-P12, pRS-P11 / pRS-P2, and NdeI-pRS-PstI fragments were ligated using Gibson ligation to form a circular plasmid pRS library containing mutations at the 32Tyr, 53Ile, 75Gln, 158Asp, and 204Lys sites.
[0043] Table 1 Primers for MjAibRS site-directed mutagenesis
[0044]
[0045] Example 2 (Construction of MjAibRS mutant library screening system)
[0046] (1) Constructing a positive screening reporter gene system plasmid
[0047] The positive selection and reporter gene system plasmid pCM-RFP is a circular plasmid composed of three fragments: pACYC184-CM112, a mutant tyrosine succinate-repressed tRNACUA expression cassette, and a 2Ala succinate-stop codon-repressed red fluorescent protein RFP expression cassette. The required primers are shown in Table 2.
[0048] Table 2 Primer list for the screening system
[0049]
[0050] pACYC184-CM112 originates from the pACYC184 vector, and a succinate stop codon mutation occurs at the 112Asp position of pACYC184 chloramphenicol acetyltransferase (CAT). The specific origin is as follows:
[0051] Using pACYC184 as a template, fragments of 3334 bp and 925 bp were amplified using primer pairs pCM-RFP-P1 / P2 and pCM-RFP-P3 / P4, respectively. The fragments were then separated and recovered by agarose gel electrophoresis. Using the recovered two fragments as templates, a 4245 bp fragment was amplified using pCM-RFP-P2 / P3 as primers. The fragment was then separated and recovered by agarose gel electrophoresis to obtain the pACYC184-CM112 fragment. The amino acid sequence of the CAT mutation at position 112Asp is SEQ ID No. 18, and the gene sequence of pACYC184-CM112 is SEQ ID No. 19.
[0052] The mutant tyrosine amber repressor tRNACUA is a mutant tyrosine amber repressor tRNA derived from *Methanococcus janaschii* (MjtRNATyr) under the control of the proteinase K (ProK) promoter and ProK terminator. The gene coding sequence is SEQ ID No. 20, where 1-62 are the ProK promoter sequence, 63-139 are the mutant tyrosine amber repressor tRNACUA, and 140-174 are the ProK terminator. This sequence was fully synthesized by Suzhou Genewiz Biotechnology Co., Ltd. Using the synthesized gene as a template, a 174 bp fragment was amplified using primers pCM-RFP-P5 / P6, and the tRNACUA expression cassette was separated and recovered by agarose gel electrophoresis.
[0053] The amino acid sequence of the red fluorescent protein RFP, suppressed by the 2Ala amber stop codon, is SEQ ID No. 21, and the encoding gene sequence is SEQ ID No. 22. This sequence was fully synthesized by Suzhou Genewise Biotechnology Co., Ltd. Using the synthesized gene as a template, a 709 bp fragment was amplified using primers pCM-RFP-P7 / P8, and the fragment was separated and recovered by agarose gel electrophoresis. The recovered fragment was ligated to a vector double-digested with pBAD24 NheI and HindIII using the Gibson ligation method. Using the ligated plasmid as a template, a 2426 bp fragment was amplified using primers pCM-RFP-P9 / P10, and the fragment was separated and recovered by agarose gel electrophoresis to obtain the expression cassette of the red fluorescent protein RFP suppressed by the 2Ala amber stop codon, controlled by the L-arabinose promoter and the rrnB terminator.
[0054] The linear sequence pACYC184-CM112, the tRNACUA expression cassette, and the expression cassette of the red fluorescent protein RFP suppressed by the 2Ala amber stop codon were ligated into a circular plasmid pCM-RFP using the Gibson ligation method. The vector map is shown below. Figure 2 The vector sequence is SEQ ID No. 45. pCM-RFP was transformed into BL21DE3, and positive transformants BL21DE3-pCM-RFP were screened using 12.5 μg / mL Tet (tetracycline).
[0055] (2) Construction of negative screening reporter gene system plasmid pCM-Bar
[0056] The negative selection and reporter gene system plasmid pCM-Bar is a circular plasmid composed of three fragments: pACYC184-CM112, a mutant tyrosine succinate repressor tRNACUA expression cassette, and a Barnase expression cassette repressed by succinate stop codons at positions 2Gln, 44Asp, and 65Gly. The required primers are shown in Table 2.
[0057] A barnase repressed by succinate stop codons at positions 2Gln, 44Asp, and 65Gly, with the amino acid sequence SEQ ID No. 23 and the encoding gene sequence SEQ ID No. 24, was synthesized by Suzhou Genewise Biotechnology Co., Ltd. Using the synthesized gene as a template, a 366 bp fragment was amplified using primers pCM-Bar-P1 / P2, and the fragment was separated and recovered by agarose gel electrophoresis. The recovered fragment was ligated to a vector double-digested with pBAD24 NheI and HindIII using the Gibson ligation method. Using the ligated plasmid as a template, a 2081 bp fragment was amplified using primers pCM-RFP-P9 / P10, and the fragment was separated and recovered by agarose gel electrophoresis to obtain the expression cassette of barnase repressed by succinate stop codons at positions 2Gln, 44Asp, and 65Gly, controlled by an L-arabinose promoter and an rrnB terminator.
[0058] The linear sequence pACYC184-CM112, the tRNACUA expression cassette, and the barnase expression cassette suppressed by 2Gln, 44Asp, and 65Gly amber stop codons were ligated into a circular plasmid pCM-BAR using the Gibson ligation method. The vector map is shown below. Figure 3 The vector sequence is SEQ ID No. 46. pCM-BAR was transformed into BL21DE3, and positive transformants BL21DE3-pCM-BAR were screened using 12.5 μg / mL Tet.
[0059] Example 3 (Screening of the MjAibRS mutant library)
[0060] To identify active MjAibRS variants from the mutant MjAibRS library, positive screening was performed using the CAT and RFP reporting systems, and negative screening was performed using the Barnase reporting system. The specific screening methods are as follows:
[0061] (1) Positive screening
[0062] Pick E. coli BL21DE3 / pCM-RFP into 5 mL of LB broth containing 12.5 μg / mL LTet and incubate overnight at 37 °C. Expand the culture 1:100 by dilution in 80 mL of LB broth containing 12.5 μg / mL LTet and incubate at 37 °C for 2–3 h until OD (dose expiratory rate). 600 The bacterial culture was collected after reaching a pH of 0.4-0.6. The culture was pre-cooled for 10 min, centrifuged at 4 °C to collect the cells, resuspended in pre-cooled 10% glycerol, centrifuged again at 4 °C, and the process was repeated at least once. Finally, the cells were resuspended in 400 μL of 10% glycerol, aliquoted into 100 μL tubes, flash-frozen in liquid nitrogen, and stored at -80 °C. The plasmid of the MjAibRS mutant library constructed in Example 1 was electroporated into E. coli BL21DE3 / pCM-RFP. After recovery at 37 °C and 220 rpm for 45 min, the bacterial culture was plated onto plates containing 100 μg / mL Amp (ampicillin), 12.5 μg / mL Tet, 80 μg / mL Cm (chloramphenicol), and 1 mM Aib (Amp and Tet are selection markers for pRS and pCM-RFP, respectively). Surviving strains indicate that the MjAibRS mutant within the bacteria can recognize Aib or any native amino acid to aminoacylate tRNACUA. The MjAibRS mutant plasmid pRSP1 was extracted from surviving cells.
[0063] (2) Negative screening
[0064] E. coli BL21DE3 / pCM-BAR electrotransfer competent cells were prepared. The MjAibRS mutant plasmid pRSP extracted in (1) was electrotransformed into E. coli BL21DE3 / pCM-BAR. After recovery at 37 ℃ and 220 rpm for 45 min, the bacterial culture was plated on plates containing 100 μg / mL Amp, 12.5 μg / mL Tet, and 0.2% L-arabinose. The expression of the toxic protein Barnase was initiated under the control of the L-arabinose operon. If the MjAibRS mutant in the cells could recognize any natural amino acid and synthesize the toxic protein Barnase, the cells would be lethal. Surviving strains indicated that the MjAibRS mutant in the cells could not recognize any natural amino acid and cause tRNACUA aminoacylation. The MjAibRS mutant plasmid pRSN1 was extracted from the surviving cells.
[0065] (3) Repeated screening and finalization
[0066] Repeat the operations in (1) and (2) of this embodiment, and perform a new round of positive and negative screening on the MjAibRS mutant plasmid pRSN extracted in (2) to obtain pRSP2 and pRSN2. Electroporate pRSN2 into E. coli BL21DE3 / pCM-RFP to obtain E. coli BL21DE3 / pCM-RFP / pRSN2, and dilute it 10 at 37°C. 5 The solutions were prepared on plates containing 100 μg / mL Amp, 12.5 μg / mL LTet, 80 μg / mL Cm, 1 mM Aib, and 0.2% L-arabinose. Aib-dependent chloramphenicol resistance and Aib-dependent RFP fluorescence were then detected in 100 selected clones.
[0067] (4) Detection of Aib-dependent chloramphenicol resistance
[0068] Set up the following 6 groups of tablets:
[0069] The first set of plates was supplemented with 100 μg / mL Amp and 12.5 μg / mL Tet;
[0070] The second set of plates was supplemented with 100 μg / mL Amp and 10 μg / mL Tet.
[0071] The third set of plates was supplemented with 100 μg / mL Amp, 12.5 μg / mL Tet, and 100 μg / mL Cm;
[0072] The fourth set of plates was supplemented with 100 μg / mL Amp, 12.5 μg / mL Tet, and 1 mM Aib;
[0073] The fifth group of plates was supplemented with 100 μg / mL Amp, 12.5 μg / mL Tet, 10 μg / mL Cm, and 1 mM Aib;
[0074] The sixth plate was supplemented with 100 μg / mL Amp, 12.5 μg / mL LTet, 100 μg / mL Cm, and 1 mM Aib.
[0075] The results showed that bacteria with added Aib could grow on high-concentration Cm plates, while bacteria without added Aib showed almost no growth on plates with 10 μg / mL Cm. Therefore, the significant difference in Cm resistance with and without Aib indicates that the selected MjAibRS / tRNACUA strain is specific for Aib insertion into the amber codon in 20 natural amino acids of the bacteria.
[0076] (5) Aib-dependent RFP fluorescence detection
[0077] Two plates were prepared. One plate was prepared with 100 μg / mL Amp, 12.5 μg / mL Tet, 80 μg / mL Cm, and 0.2% L-arabinose. The second plate was prepared with 100 μg / mL Amp, 12.5 μg / mL Tet, 80 μg / mL Cm, 0.2% L-arabinose, and 1 mM Aib.
[0078] The results showed that bacteria with added Aib produced red fluorescence, while bacteria without added Aib did not produce red fluorescence (see...). Figure 4 The significant difference between the presence and absence of Aib demonstrates specificity for inserting Aib into all 20 naturally occurring amino acids found in the cell, within the selected synthase tRNA pairs.
[0079] (6) Sequencing
[0080] pRS plasmids were extracted from the selected bacterial cells and sent for sequencing. Based on the sequencing results, five MjAibRS plasmids were obtained, namely MjAibRS1, MjAibRS2, MjAibRS3, MjAibRS4, MjAibRS5, and MjAibRS6. Their protein sequences are shown in SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, and SEQ ID NO.42, respectively. The mutation site characteristics are shown in Table 3.
[0081] Table 3. Sequences of selected synthases
[0082]
[0083] (7) EGFP verification
[0084] Green fluorescent protein (EGFP) was used as the target protein for modification to verify the introduction of Aib. The target sites for Aib modification were 2Val and 30Ser. The target EGFP protein was expressed using pEvol-EGFPTAG. The EGFP amino acid sequence suppressed by the amber stop codon at 2Val and 30Ser is SEQ ID No. 43. A 6*His tag was inserted at the N-terminus, and the encoding gene sequence is SEQ ID No. 44. This sequence was fully synthesized by Suzhou Genewise Biotechnology Co., Ltd. The pEvol-pAzF vector was double-digested with BglII and SalI, and the linearized vector pEvol-BglII-SalI was recovered after gel digestion. The synthesized gene was double-digested with BglII and SalI, and the fragment EGFPTAG-BglII-SalI was recovered after gel digestion. The recovered fragment was ligated into the double-digested pEvol-BglII-SalI vector using the T4 ligation method to obtain the plasmid pEvol-EGFPTAG.
[0085] The pEvol-EGFPTAG vector was double-digested with NdeI and PstI, and the linearized vector pEvol-EGFPTAG-NdeI-PstI was recovered after gel digestion. The six selected MjAibRS plasmids pRS were double-digested with NdeI and PstI, and the fragments MjAibRS1-NdeI-PstI, MjAibRS2-NdeI-PstI, MjAibRS3-NdeI-PstI, MjAibRS4-NdeI-PstI, MjAibRS5-NdeI-PstI, and MjAibRS6-NdeI-PstI were recovered after gel digestion. These recovered fragments were then combined with the linearized vector pEvol-EGFPTAG-NdeI-PstI. The plasmids pEvol-EGFPTAG-NdeI-PstI were obtained by ligation using the T4 ligation method: pEvol-EGFPTAG-MjAibRS1, pEvol-EGFPTAG-MjAibRS2, pEvol-EGFPTAG-MjAibRS3, pEvol-EGFPTAG-MjAibRS4, pEvol-EGFPTAG-MjAibRS5, and pEvol-EGFPTAG-MjAibRS6.
[0086] The above 6 plasmids were transformed into E. coli BL21DE3 to obtain transformants, which were then expanded into liquid medium to OD. 600 At approximately 0.4–0.6, the mice were divided into two groups: one group was induced with 0.2% L-arabinose, and the other group was induced with 0.2% L-arabinose and 1 mM Aib.
[0087] The relative fluorescence values of EGFP in E. coli with and without the addition of Aib (see...) Figure 5 )as follows:
[0088] MjAibRS1: 0.12×10 6 and 1.55×10 6 ;
[0089] MjAibRS2: 0.15×10 6 and 1.37×10 6 ;
[0090] MjAibRS3: 0.16×10 6 and 1.31×10 6 ;
[0091] MjAibRS4: 0.13×10 6 and 1.24×10 6 ;
[0092] MjAibRS5: 0.15×10 6 and 1.19×10 6 ;
[0093] MjAibRS6: 0.13×10 6 and 1.34×10 6 .
[0094] Bacterial cells containing the pEvol-EGFPTAG-MjAibRS1 plasmid, both with and without Aib, were cultured separately. EGFP in the lysate was purified using a nickel column. SDS-PAGE analysis showed that the cell lysate with Aib could be purified using a nickel column for EGFP protein, while the cell lysate without Aib could not. This indicates that the green fluorescent protein produced by cells without Aib is not full-length. Transforming MjAibRS1 into BL21DE3 yields a single Aib-modified EGFP protein.
[0095] Example 4 (Biosynthesis of Aib-modified smegglutinin intermediate)
[0096] The 2-methylalanine (Aib) translation systems MjTyrRS, MjAibRS1, MjAibRS2, MjAibRS3, MjAibRS4, and MjAibRS5 selected in Example 3 were used in the biosynthesis of smegglutinin intermediates containing the non-natural amino acid 2-methylalanine (Aib). The amino acid sequence of the smegglutinin intermediate is shown in SEQ ID No. 47, specifically H*EGTFTSDVSSYLEGQAAKEFIAWLVRGRG*. Its nucleotide sequence is SEQ ID No. 48. This sequence was totally synthesized by Suzhou Genewiz Biotechnology Co., Ltd., with KpnI and EcoRI restriction sites added to both ends, respectively.
[0097] The pET32b-kan vector was double-digested with KpnI and EcoRI, and the linearized vector pET32b-kan-KpnI-EcoRI was obtained after gel digestion. The synthesized gene was double-digested with KpnI and EcoRI, and the fragment obtained after gel digestion was ligated into the double-digested pET32b-kan-KpnI-EcoRI vector using the T4 ligation method to obtain the plasmid pET32b-kan-SEAib. The correctly sequenced plasmid was transformed into BL21DE3-pEvol-EGFPTAG-MjAibRS1, and the fusion protein TrxA-SEAib was induced to express using IPTG. Bacterial cells were collected, sonicated, and the results are shown in Table 4. The SDS-PAGE results for cell number 2 are shown in Table 4. Figure 6 This demonstrates the successful expression of the Aib-modified smegglutinin intermediate.
[0098] Table 4
[0099]
[0100] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.
Claims
1. A mutant aminoacyl-tRNA synthetase, characterized in that, The aminoacyl-tRNA synthetase mutant is any one of the following mutations in the amino acid sequence of the wild-type tyrosine-tRNA synthetase from *Methanococcus japonicus*: 1) Form 1: Replace position 32 with glycine, position 53 with alanine, position 75 with glutamic acid, position 158 with threonine, and position 204 with arginine; 2) Form 2: 32 is replaced with glutamine, 53 with alanine, 75 with lysine, 158 with glutamic acid, and 204 with serine; 3) Form 3: Replace position 32 with glycine, position 53 with aspartic acid, position 75 with arginine, position 158 with glutamic acid, and position 204 with serine; 4) Form 4: 32 is replaced with glutamine, 53 with alanine, 75 with lysine, 158 with threonine, and 204 with arginine; 5) Form 5: Replace position 32 with glycine, position 53 with aspartic acid, position 75 with glutamic acid, position 158 with threonine, and position 204 with serine; 6) Form 6: Replace position 32 with glycine, position 53 with valine, position 75 with glutamic acid, position 158 with threonine, and position 204 with arginine.
2. The aminoacyl-tRNA synthetase mutant according to claim 1, characterized in that, The amino acid sequence of the aminoacyl-tRNA synthetase mutant is shown in SEQ ID No. 37-42.
3. The aminoacyl-tRNA synthetase mutant according to claim 1, characterized in that, The amino acid sequence of the wild-type tyrosine-tRNA synthetase from Methanococcus japonicus is shown in SEQ ID No.
2.
4. A gene encoding the aminoacyl-tRNA synthetase mutant of claim 1.
5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the gene as described in claim 4.
6. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria comprises the recombinant expression vector as described in claim 5.
7. Use of the aminoacyl-tRNA synthetase mutant according to any one of claims 1-3 in the preparation of recombinant proteins containing non-natural amino acids.
8. The application according to claim 7, characterized in that, The non-natural amino acid is 2-aminoisobutyric acid.
9. The application according to claim 7, characterized in that, The recombinant protein is selected from any one of smegglutinin, tasmegglutinin, abaloidin, and membrane-penetrating peptides.