Archaeal tyrosyl-tRNA synthetase mutants and their applications

Abstract

The present invention discloses an archaeal tyrosyl-tRNA synthetase mutant and its application, which is obtained by mutation of the amino acid sequence shown in SEQ ID NO.12 at positions 283-286. The present invention modifies the sequence of the tRNA binding region of pCNPheRS to improve the recognition rate of tRNA, thereby improving the introduction efficiency of non-natural amino acids. By introducing non-natural amino acids into the expression of GFP fluorescent protein, it can be seen that the mutants TSDV, TPAL and KPAL all express more GFP protein containing non-natural amino acids than the unmutated pCNPheRS, that is, the efficiency of introducing non-natural amino acids is high. And the three mutants have improved the introduction efficiency of eight non-natural amino acids. It has been verified that TSDV can introduce pAcF into Taq DNA polymerase, and the expression amount can be used for subsequent activity studies. The present invention solves the problem of low efficiency of non-natural amino acid introduction and greatly expands the research on the structure and function of proteins.

Description

Technical Field

[0001] The present invention relates to the field of enzyme engineering technology, in particular to an archaeal tyrosyl-tRNA synthetase mutant and an application thereof. Background Art

[0002] Enzymes, as important biocatalysts, offer advantages such as high catalytic efficiency, strong specificity, and mild reaction conditions. Consequently, they are widely used in medicine, analytical testing, industrial and agricultural production, and other fields. However, enzymes also suffer from shortcomings such as poor thermal stability, limited substrate compatibility, poor tolerance to organic solvents, and a limited range of reactions that can be catalyzed by enzymes. These shortcomings limit their application in large-scale industrial production. The rise of directed evolution technology has enabled the modification and evolution of enzyme properties, significantly improving these issues and promoting the application of enzymes in compound synthesis.

[0003] Currently, methods such as gene point mutation, combinatorial mutagenesis, computer-assisted protein design, and chemical modification can impart new functions to proteins. However, these methods rely on the 20 natural amino acids themselves, which contain only a few functional groups such as amino, hydroxyl, sulfhydryl, and carboxyl groups, resulting in limited functionality. Non-natural amino acids (ncAAs) contain a variety of groups, including acetyl, alkyne, nitro, halide, azide, aldehyde, and ketone groups, and can undergo a variety of modification reactions, such as glycosylation, photochemical reactions, and fluorescence display. This can greatly expand the structure and function of proteins. Furthermore, ncAAs with active groups have broad applications in protein structure research, protein function regulation, the construction of novel biomaterials, and pharmaceutical research and development.

[0004] A commonly used method for introducing ncAAs is genetic code expansion (GCE) technology, the core of which is aminoacyl-tRNA synthetase (aaRS), which can incorporate ncAAs into proteins on the ribosome, allowing the controllable introduction of ncAAs during protein synthesis. The key to GCE technology is the one-to-one correspondence between the ncAA corresponding to a specific codon and the orthogonal tRNA / aaRS. In vivo codon expansion requires three conditions: (1) the specific introduction of a blank codon that is not used to encode other amino acids at the desired site, usually the amber stop codon UAG; (2) an orthogonal tRNA that is not recognized by the endogenous aaRS and can recognize and encode the blank codon during translation; (3) an aminoacyl-tRNA synthetase that does not interact with endogenous tRNA and is modified to interact with ncAA, thereby recognizing and introducing ncAA instead of endogenous natural amino acids.

[0005] Over the past decade, GCE-based technology for introducing non-natural amino acids into proteins has rapidly developed and been applied. Its applications in the field of therapeutic proteins include antibody-drug conjugates (ADCs), bispecific antibodies (BsAbs), immunotherapy, therapeutic vaccines, long-acting protein therapies, modification of therapeutic proteins, and other therapeutic-related applications. Furthermore, ncAAs can be used as probes to study protein structure and function. For example, some photocontrollable ncAAs, such as o-nitrobenzyl-L-tyrosine (ONBY), undergo decomposition reactions to form similar natural amino acids upon exposure to light of a certain wavelength. This property allows these ncAAs to serve as light-responsive elements, enabling photocontrol of enzyme function. Furthermore, the introduction of ncAAs into enzymes can improve their catalytic activity, stereoselectivity, and thermal stability.

[0006] Although genetic codon expansion technology has been successfully applied, the incorporation efficiency of most existing unnatural amino acids is low. This is primarily due to the inefficiency of modified aminoacyl-tRNA synthetases in catalyzing the aminoacylation reaction between tRNA and the corresponding ncAA. This results in low expression levels of recombinant proteins containing ncAAs, significantly limiting their application in protein structure and function research and modification, and increasing the cost of industrial applications. Therefore, modifying tRNA / aminoacyl-tRNA synthetases to enhance the efficiency of ncAA incorporation into target proteins and increase the expression of recombinant proteins containing ncAAs is crucial for both basic research and industrial production. Summary of the Invention

[0007] To address the problem of low efficiency in the introduction of unnatural amino acids, the present invention provides a site-directed mutagenesis-modified archaeal tyrosyl-tRNA synthetase (MjTyrRS) mutant that can efficiently introduce multiple unnatural amino acids into target proteins.

[0008] The specific technical solutions are as follows:

[0009] A tyrosyl-tRNA synthetase mutant is obtained by mutating a wild-type tyrosyl-tRNA synthetase from Methanococcus jannaschii (M. jannaschii), wherein the specific mutation is any one of the following:

[0010] (1) Histidine at position 283 mutated to threonine, proline at position 284 mutated to serine, methionine at position 285 mutated to aspartic acid, and aspartic acid at position 286 mutated to valine;

[0011] (2) histidine at position 283 mutated to threonine, methionine at position 285 mutated to alanine, and aspartic acid at position 286 mutated to leucine;

[0012] (3) The histidine at position 283 was mutated to lysine, the methionine at position 285 was mutated to alanine, and the aspartic acid at position 286 was mutated to leucine.

[0013] Preferably, the amino acid sequence of the wild-type tyrosyl-tRNA synthetase is shown as SEQ ID NO.12.

[0014] The present invention modifies the tRNA binding region of the tyrosyl-tRNA synthetase pCNPheRS from M. jannaschii and performs saturation mutagenesis on three sites, 283, 285, and 286, which are close to the tRNA C34 (formerly G34) site. pCNPheRS is derived from MjTyrRS by modifying the catalytic region, which improves the efficiency of incorporating unnatural amino acids. Furthermore, site-directed mutagenesis at sites 283-286 was performed to construct and obtain mutants with enhanced activity, enabling the incorporation of multiple ncAAs into the target protein.

[0015] The present invention also provides a gene encoding the tyrosyl-tRNA synthetase mutant.

[0016] The present invention also provides a recombinant vector comprising the encoding gene.

[0017] The present invention also provides a genetically engineered bacterium comprising the encoding gene.

[0018] The present invention also provides the use of the tyrosyl-tRNA synthetase mutant or the encoding gene in introducing non-natural amino acids into target proteins.

[0019] Furthermore, the application includes: co-transforming the recombinant vector and the target protein plasmid into competent cells for induction culture to obtain the target protein containing non-natural amino acids.

[0020] Furthermore, the competent cells are Escherichia coli BL21 (DE3).

[0021] Furthermore, the non-natural amino acid is at least one of 4-amino-L-phenylalanine, 4-nitro-L-phenylalanine, 3-(4-acetylphenyl)alanine, L-4-cyanophenylalanine, 4-methoxy-L-phenylalanine, L-4-fluorophenylalanine, O-tert-butyl-L-tyrosine, and O-benzyl-L-tyrosine.

[0022] The present invention also provides a method for introducing non-natural amino acids into a target protein, wherein the recombinant vector and the protein plasmid are co-transformed into competent cells for induction culture to obtain a protein containing non-natural amino acids.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention modified the tRNA binding region of pCNPheRS. Through site-directed mutagenesis at four sites and random screening of saturation mutagenesis at three sites, mutants with higher ncAA incorporation efficiency were obtained. The modified mutants were then used to attempt to introduce eight commonly used ncAAs, and the results showed improved efficiency for all of these ncAAs. Furthermore, the present invention used the unnatural amino acid 3-(4-acetylphenyl)alanine (pAcF) for large-scale expression of GFP protein, successfully producing pAcF-incorporated GFP protein. pAcF was also introduced into Taq DNA polymerase and successfully expressed for subsequent studies. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The ncAAs involved in the present invention and their structural formulas;

[0026] Among them, NC1 is 4-amino-L-phenylalanine, NC2 is 4-nitro-L-phenylalanine, NC3 is 3-(4-acetylphenyl)alanine, NC4 is L-4-cyanophenylalanine, NC5 is 4-methoxy-L-phenylalanine, NC6 is L-4-fluorophenylalanine, NC7 is O-tert-butyl-L-tyrosine, and NC8 is O-benzyl-L-tyrosine.

[0027] Figure 2 The figure shows the comparison of the introduction efficiency of 8 ncAAs by pCNPheRS and TSDV;

[0028] Wherein, Negative is a negative control without the addition of unnatural amino acids, and Positive is a full-length GFP protein without TAG mutation, i.e., a positive control.

[0029] Figure 3 Comparison of the introduction efficiency of TPAL, KPAL, and pCNPheRS for eight ncAAs;

[0030] Negative is a negative control without the addition of unnatural amino acids, and Positive is a full-length GFP protein without the amber codon mutation, i.e., a positive control.

[0031] Figure 4 This is the electrophoresis diagram of GFP protein purification in Example 3;

[0032] Among them, lane 1 is TSDV+pET21a-GFP2TAG+pAcF,

[0033] Lane 2 is TPAL+pET21a-GFP2TAG+pAcF,

[0034] Lane 3 is KPAL+pET21a-GFP2TAG+pAcF,

[0035] Lane 4 is pCNPheRS+pET21a-GFP2TAG+pAcF,

[0036] Lane 5 is the negative control of pCNPheRS+pET21a-GFP2TAG without adding unnatural amino acids.

[0037] Lane 6 is the full-length GFP protein without the TAG mutation, which is the positive control.

[0038] Figure 5 This is the electrophoresis diagram of Taq DNA polymerase purification in Example 3;

[0039] Among them, lane 1 is TSDV+pET21a-Taq671TAG+pAcF, and lane 2 is TSDV+pET21a-Taq671TAG without pAcF. DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with specific embodiments. The following are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto.

[0041] Reagents and materials: unnatural amino acids and tyrosyl-tRNA synthetase; unnatural amino acids used in the present invention (such as Figure 1 ) A total of 8 kinds, the mother liquor concentration is 100mM, and the final concentration is 1mM. The tyrosyl-tRNA synthetase involved in the present invention is from Methanococcus jannaschii (M.jannaschii), and has been transformed in the amino acid binding region to improve the introduction efficiency of non-natural amino acids pAcF. The plasmid is pCNPheRS (nucleotide sequence is shown in SEQ ID NO.1). On this basis, the tRNA binding part is transformed to obtain mutants with greater activity improvement, which are respectively named TSDV (nucleotide sequence shown in SEQ ID NO.2), TPAL (nucleotide sequence shown in SEQ ID NO.3), and KPAL (nucleotide sequence shown in SEQ ID NO.4). The target protein used is GFP fluorescent protein. The gene is synthesized by Qingke Company and connected to the pET21a vector, wherein the second site serine triplet codon sequence of GFP is mutated to an amber stop codon (TAG) (nucleotide sequence is shown in SEQ ID NO.5).

[0042] Example 1 Construction and activity detection of TSDV mutants

[0043] TSDV is a modification of the tRNA binding domain of pCNPheRS. Using site-directed mutagenesis, the amino acids HPMD at positions 283-286 of pCNPheRS were mutated to TSDV. Furthermore, the serine at the second position of the green fluorescent protein (GFP) was mutated to the amber stop codon TAG to test the efficiency of unnatural amino acid incorporation. The experimental procedure is as follows:

[0044] 1. Site-directed mutagenesis

[0045] Mutation primers were designed based on pCNPheRS (primer design website: https: / / crm.vazyme.com / cetool / multipoint.html) and synthesized by Beijing Qingke Biotechnology Co., Ltd. The obtained primers are as follows:

[0046] TSDV-F: GAATTGaccagcgatgttTTAAAAAATGCTGTAGCTGAAGAACTT;

[0047] TSDV-R:aacatcgctggtCAATTCCTTATTTTTAAATAAACTCTCTAACT.

[0048] Using the wild-type plasmid, the pCNPheRS circular plasmid, as a template, a circular PCR product was generated by polymerase chain reaction. The PCR product was digested with DpnI at 37°C for 1 hour, digesting the original template. The product was then transformed into DH5α competent cells and plated on plates containing spectinomycin (Spe) resistance. After inverting in a 37°C oven for 14-16 hours, three individual cells were picked and incubated in LB medium supplemented with Spe resistance. After incubation at 37°C on a shaker for 14-16 hours, the cells were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing and verification of the correct clone. Glycerol strains were also prepared and stored at -80°C. The same method was used to mutate the amino acid at position 2 of the synthesized pET21a-GFP2TAG back to its original serine, resulting in wild-type GFP. The plasmid for this mutation is pET21a-GFP. The primers are as follows:

[0049] wtGFP-F: GagcAAAGGAGAAGAACTTTTCACTGGAGTTG;

[0050] wtGFP-R:GTTCTTTCCTTTgctCATATGTATATCTCCTTCTTAAAGTTAAACAAA.

[0051] 2. Activate the strain and extract the plasmid

[0052] The correctly sequenced glycerol strain was streaked, and a single strain was selected for expansion. The plasmid of the mutant TSDV was extracted using a kit (SanPrep Column-Based Plasmid DNA Miniprep Kit). For details on plasmid extraction, refer to the kit's instructions. pET21a-GFP, pET21a-GFP2TAG, and pCNPheRS plasmids were also extracted.

[0053] 3. Activity Detection

[0054] pET21a-GFP+TSDV, pET21a-GFP2TAG+TSDV, and pET21a-GFP2TAG+pCNPheRS were co-transformed into competent BL21 E. coli and plated onto Amp+Spe-resistant plates. Three individual cells from each plate were placed in 5 mL of LB medium containing Amp+Spe resistance (test tubes) and incubated at 37°C, 300 rpm, and shaken for 6 h. The OD600 value was measured. Based on the OD600 value, the desired volume of bacterial culture (x μL) (x = 200 × 0.8 / OD600) was calculated and added to a 1.5 mL centrifuge tube. The tubes were centrifuged at 4000 rpm for 1 min, and the supernatant was discarded. 800 μL of premixed GMML medium (GMML+Amp+Spe+IPTG) was added to each tube, and 1 mM of different ncAAs was added to each tube. After resuspension, the cells were cultured at 37°C and 300 rpm for 11 h, and then loaded onto an ELISA plate to measure the OD600 value, and the fluorescence intensity was measured by 485 nm excitation and 515 nm emission.

[0055] Results: As Figure 2 As shown in the results, compared with pCNPheRS, TSDV has improved the introduction efficiency of 8 ncAAs, and has higher introduction efficiency for NC2, NC3, NC4, NC5 and NC7.

[0056] Table 1 Activity of pCNPheRS and TSDV against 8 ncAAs

[0057]

[0058]

[0059] Example 2 Construction and activity detection of TPAL and KPAL mutations

[0060] Aminoacyl-tRNA synthetase mainly includes two functional domains, the catalytic central domain (CCD) and the domain that binds to the tRNA anticodon (ABD). TPAL and KPAL in the present invention are saturated mutations at three sites 283, 285 and 286, which are close to the tRNA anticodon C34 (formerly G34) site, to screen mutants with improved efficiency of introducing ncAA. Single bacteria were randomly selected for sequencing, and the efficiency of 79 sequences introduced into pAcF was tested. Among them, when 283H, 285M and 286D were each mutated to T, A, L and K, A, L, the measured fluorescence value (fl / OD600) was greatly improved. At the same time, the introduction efficiency of the remaining 7 kinds of ncAAs was tested using these two mutants, and the introduction efficiency was improved. The experimental process is as follows:

[0061] Primers for saturation mutagenesis were designed based on pCNPheRS (primer design was obtained from the Vazyme website: https: / / crm.vazyme.com / cetool / multipoint.html) and synthesized by Beijing Qingke Biotechnology Co., Ltd. The primers obtained are as follows:

[0062] 28356-F:GAATTGnnkCCAnnknnkTTAAAAAATGCTGTAGCTGAAGAACTT,

[0063] 28356-R:mnnmnnTGGmnnCAATTCCTTATTTTTAAATAAACTCTCTAACT.

[0064] 1. Prepare pET21a-GFP2TAG competent cells, test the efficiency of competent cells (transfer 3-100 μL), incubate the cells for 1 hour, and take 50 μL to plate (100 + 900) μL;

[0065] 2. Perform PCR amplification using pCNPheRS plasmid (100 ng / μL) as template and 28356-F / R as primers. Prepare PCR premix according to Table 2.

[0066] Table 2 PCR premix

[0067]

[0068]

[0069] The entire preparation process was performed on ice. The reaction conditions were preheating at 95°C for 3 minutes, followed by 30 cycles of melting at 95°C for 30 seconds, annealing at 58°C for 45 seconds, and extension at 72°C for 2 minutes. Finally, a final extension at 72°C for 5 minutes was performed. After PCR, electrophoresis (130V for 30 minutes) was performed to observe the presence of the desired band.

[0070] 3. Add 1 μL of DpnI enzyme to the PCR product and digest it at 37°C for 1 hour to digest the original template;

[0071] 4. Transform the DpnI digestion product into pET21a-GFP2TAG competent cells. Co-transform this digestion product and pET21a-GFP into BL21 competent cells as a positive control (transform 3 μL of each product into 100 μL competent cells). Incubate the bacteria for 1 hour. Take 100 μL and apply it to Amp+Spe resistance plates.

[0072] 5. Pick 96 individual bacteria into a 96-well plate (800 μL LB medium containing Amp+Spe resistance) and culture overnight at 37°C, 300 rpm for 15 h;

[0073] 6. Take another deep-well plate and store the glycerol bacteria (200 μL of bacterial solution plus 200 μL of 50% glycerol (v / v) at -80°C). Send the remaining bacteria to GeneWeizhi for sequencing.

[0074] 7. After the sequencing results are returned, the glycerol stock (8 μL) with the sequence obtained was inoculated into 800 μL of LB medium containing Amp + Spe resistance in a deep-well plate for expansion. Incubate at 300 rpm and 37°C for 4 h. When the bacterial liquid grows to an OD value of approximately 0.4 (approximately 11 h), centrifuge at 4000 rpm for 2 min and discard the supernatant.

[0075] 8. Add 800 μL of premixed GMML medium (GMML+Amp+Sp+IPTG+pAcF) to each tube, resuspend, and continue to culture at 37°C and 300 rpm for 11 h. Then load the sample onto an ELISA plate to measure the OD600 value and the fluorescence intensity by 485 nm excitation and 515 nm emission.

[0076] 9. The obtained TPAL and KPAL with higher activity were tested for the efficiency of introducing other ncAAs using the same activity detection method.

[0077] Results: As Figure 3 The results show that the efficiency of TPAL and KPAL in introducing the eight ncAAs is higher than that of pCNPheRS. Similarly, the introduction efficiency of NC2, NC3, NC4, NC5 and NC7 is higher.

[0078] Table 3 Activity of TPAL, KPAL and pCNPheRS against 8 ncAAs

[0079]

[0080]

[0081] Example 3 Expression and Purification of GFP Protein / Taq DNA Polymerase Introducing Unnatural Amino Acids

[0082] The present invention uses TSDV, TPAL, KPAL and pCNPheRS to introduce the unnatural amino acid pAcF at the second amino acid site of GFP, and after induction expression, the GFP protein introduced with pAcF is purified (e.g. Figure 4 ), and from the expression of GFP protein, it can be seen that the effects of TSDV, TPAL and KPAL introduced into pAcF are better than pCNPheRS, and the introduction efficiency is increased by about 2 times. In addition, the present invention uses TSDV to introduce pAcF into the 671st site of Taq DNA polymerase (such as Figure 5 ), successfully replaced the tyrosine at this site with p-acetylphenylalanine to study the catalytic mechanism of Taq DNA polymerase.

[0083] The expression and purification methods are as follows:

[0084] 1. Transform plasmids TSDV, pCNPheRS, TPAL, and KPAL into BL21 competent cells containing pET21a-GFP2TAG, respectively. Co-transform the enzymatic hydrolyzate and pET21a-GFP into BL21 competent cells. Centrifuge at 4000 rpm for 2 min, discard the supernatant, and spread 100 μL onto Amp+Spe resistance plates. Incubate inverted at 37°C overnight.

[0085] 2. Pick a single colony and transfer it to 5 mL of LB medium containing Amp+Spe resistance, and culture overnight at 37°C and 220 rpm;

[0086] 3. Transfer 1 mL of each culture to 100 mL of LB liquid medium containing Amp+Spe resistance. Incubate at 37°C, 220 rpm for 3 hours. Then add IPTG (final concentration 0.5 mM) and pAcF (final concentration 1 mM). No pAcF was added to the control group, and GFP was used as a positive control. Continue induction culture at 18°C, 220 rpm for 18 hours.

[0087] 4. Harvest the cells by centrifugation at 4000 rpm for 20 min. Resuspend the cells in 50 mM Tris-HCl buffer and place on ice for 2-3 min. Disrupt the cells using an ultrasonic disruptor.

[0088] 5. Centrifuge at 4000 rpm for 30 min, collect the supernatant, and pass it through a membrane for later use;

[0089] 6. Ni-NTA affinity column purification: This includes equilibration, sample loading, washing, and elution. First, equilibrate the column with 20 mL of Lysis Buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). Then, pass the supernatant from step 5 through the column, slowly and repeatedly loading the sample. Then, wash away impurities with 20 mL of Wash Buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0). Finally, elute the target protein with 3 mL of Elution Buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) and collect it in a centrifuge tube.

[0090] 7. Take 40 μL of the collected protein, add 10 μL of SDS-PAGE protein loading buffer, mix well and heat at 100°C for 10 minutes. Load 10 μL into each well and run the protein gel (160V, 55 minutes). Then stain with Coomassie Brilliant Blue for 2 minutes (heat and stain in a microwave oven), decolorize with tap water and take pictures.

[0091] Results: As Figure 4 As can be seen, under the same culture conditions, lanes 1, 2, 3, and 4 all have wider bands than the negative control lane 5, indicating that the unnatural amino acid was successfully incorporated into the GFP protein. Furthermore, lanes 1, 2, and 3 have wider bands than lane 4, indicating that the mutants TSDV, TPAL, and KPAL all produce higher yields of GFP protein containing pAcF than the unmutated pCNPheRS, indicating a higher efficiency of pAcF incorporation. The brightness of the GFP fluorescent protein in the centrifuge tube also indicates that all three mutants have a higher efficiency of pAcF incorporation.

[0092] like Figure 5 It can be seen that under the same culture conditions, the lane 1 is wider than the lane 2, indicating that the unnatural amino acid was successfully introduced into the Taq DNA polymerase, and the expression level of the protein containing the unnatural amino acid was significantly higher than that of the negative control without the addition of the unnatural amino acid.

Claims

1. An archaeal tyrosyl-tRNA synthetase mutant, characterized in that from Methanococcus jannaschii ( Methanocaldococcus jannaschii ) is obtained by mutation of the wild-type tyrosyl-tRNA synthetase, wherein the specific mutation is any one of the following: (1) Histidine at position 283 mutated to threonine, methionine at position 285 mutated to alanine, and aspartic acid at position 286 mutated to leucine; (2) Histidine at position 283 mutated to lysine, methionine at position 285 mutated to alanine, and aspartic acid at position 286 mutated to leucine; The amino acid sequence of the wild-type tyrosyl-tRNA synthetase is shown in SEQ ID NO.

12.

2. The gene encoding the archaeal tyrosyl-tRNA synthetase mutant according to claim 1.

3. A recombinant vector comprising the coding gene according to claim 2.

4. A genetically engineered bacterium comprising the coding gene according to claim 2.

5. Use of the archaeal tyrosyl-tRNA synthetase mutant according to claim 1 or the encoding gene according to claim 2 in introducing unnatural amino acids into a target protein.

6. The use according to claim 5, characterized in that include: The recombinant vector according to claim 3 and the target protein plasmid are co-transformed into competent cells for induction culture to obtain the target protein containing unnatural amino acids.

7. The use according to claim 6, characterized in that The competent cells are Escherichia coli BL21 (DE3).

8. The use according to claim 5, characterized in that The unnatural amino acid is at least one of the following: 4-amino-L-phenylalanine, 4-nitro-L-phenylalanine, 3-(4-acetylphenyl)alanine, L-4-cyanophenylalanine, 4-methoxy-L-phenylalanine, L-4-fluorophenylalanine, O-tert-butyl-L-tyrosine, and O-benzyl-L-tyrosine.

9. A method for introducing unnatural amino acids into a target protein, characterized in that: The recombinant vector according to claim 3 and the target protein plasmid are co-transformed into competent cells for induction culture to obtain a protein containing unnatural amino acids.

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