Methods for introducing alpha-hydroxy acids into proteins

Abstract

The present invention provides a method for introducing α-hydroxy acid into protein, comprising: (1) constructing a recombinant plasmid and a target protein plasmid; the recombinant plasmid contains a gene encoding a mutant aminoacyl-tRNA synthetase; the target protein plasmid contains a gene encoding a target protein, and the site where the target protein plasmid needs to introduce α-hydroxy acid is mutated to a stop codon TAG; (2) co-transforming the recombinant plasmid and the target protein plasmid into a host cell, inducing and culturing the host cell, and adding α-hydroxy acid at the same time. The present invention obtains a method for effectively introducing α-hydroxy acid into protein by applying a mutant aminoacyl-tRNA synthetase capable of recognizing α-hydroxy acid; the method uses fluorescent protein EGFP as a reporter gene, the 149th amino acid base sequence of the EGFP is mutated to a stop codon TAG, and the EGFP can be used to detect the introduction efficiency of α-hydroxy acid, and can be applied to all detections of α-hydroxy acid introduction.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical engineering, and in particular to a method for introducing alpha-hydroxy acid into protein. Background Art

[0002] Like unnatural amino acids, α-hydroxy acids cannot be recognized and introduced by the organism's own aminoacyl tRNA synthetase. For proteins, especially peptide drugs, the modification of α-hydroxy acids may not only enhance the efficacy of peptide drugs and reduce drug toxicity, but also greatly reduce the immunogenicity of peptide drugs and reduce immune rejection due to the incorporation of α-hydroxy acids. It is also expected that through modification, peptide drugs can be "carried" with other chemical attachments, thereby leading to the emergence of new methods for treating diseases. For the modification of peptide drugs, most of them are modified only by chemical synthesis.

[0003] Therefore, there is an urgent need in the art to develop a method for site-specifically introducing different α-hydroxy acids into proteins. Summary of the invention

[0004] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a method for introducing α-hydroxy acid into protein, so as to solve the problem that α-hydroxy acid cannot be directly introduced into protein in the prior art. At the same time, the present invention will also provide a plasmid system for introducing a predetermined α-hydroxy acid into a target protein or preparing a target protein containing α-hydroxy acid.

[0005] In order to achieve the above-mentioned object and other related objects, the present invention provides the following technical solutions:

[0006] The first aspect of the present invention provides a method for introducing α-hydroxy acid into protein, comprising the following steps:

[0007] (1) Construction of recombinant plasmid and target protein plasmid;

[0008] The recombinant plasmid contains a gene encoding a mutant aminoacyl-tRNA synthetase;

[0009] The target protein plasmid contains a gene encoding the target protein, and the site where the α-hydroxy acid is to be introduced into the target protein plasmid is mutated to a stop codon TAG;

[0010] (2) The recombinant plasmid and the target protein plasmid are co-transformed into host cells, and the host cells are induced and cultured while adding α-hydroxy acid.

[0011] The term "α-hydroxy acid" as used herein has a well-known meaning in the art, and refers to a compound having both a hydroxyl group and a carboxyl group on the α-carbon, and its structure is shown in formula (I):

[0012] (I);

[0013] Wherein, R can be any group, including but not limited to the side chain of natural amino acids.

[0014] In a preferred embodiment, the α-hydroxy acid includes any one of 2-hydroxyheptanoic acid, S-allylcysteic acid, and S-tert-butylcysteic acid.

[0015] In a preferred embodiment, the mutant aminoacyl-tRNA synthetase is mutated corresponding to the wild-type aminoacyl-tRNA synthetase of Methanosarcina mazei (MmPylRS), and the mutation is as follows:

[0016] N346M / C348W / Y384F;

[0017] or N346M / C348W / Y384F / V401A;

[0018] or N346M / C348W / Y384F / V401K;

[0019] or N346M / C348W / Y384F / W417H;

[0020] or N346M / C348W / Y384F / V401A / W417H;

[0021] The amino acid sequence of the wild-type Methanosarcina mazei aminoacyl-tRNA synthetase (MmPylRS) is shown in SEQ ID NO.1.

[0022] In a preferred example, the nucleotide sequence encoding the wild-type MmPylRS is shown in SEQ ID NO.2.

[0023] In a preferred embodiment, the recombinant plasmid is pEvolve-MmRStRNA. The target protein is any protein that needs to introduce α-hydroxy acid.

[0024] In a preferred example, the recombinant plasmid / target protein plasmid further comprises one or more of a resistance gene, a tag sequence, a repressor protein gene (araC), and a promoter gene (araBAD).

[0025] Wherein, the resistance gene is selected from one or more of the ampicillin resistance gene (AmpR), the chloramphenicol resistance gene (CmR), the kanamycin resistance gene (KanaR), and the tetracycline resistance gene (TetR).

[0026] In a preferred example, the host cell includes Escherichia coli (E. coli); preferably E. coli DH10B.

[0027] In a preferred example, when the Escherichia coli is cultured, the culture temperature is 37°C, the inducer includes arabinose, and the concentration of α-hydroxy acid is 1%.

[0028] In a preferred embodiment, EGFP is used as a reporter protein to detect the introduction efficiency of α-hydroxy acid. The 149th amino acid base sequence of EGFP is mutated to the stop codon TAG, and a reporter protein plasmid pBAD-EGFP (149TAG) is constructed, and the reporter protein plasmid contains a gene encoding the reporter protein. The present invention uses the enhanced green fluorescent protein gene (EGFP) as a reporter protein to detect the introduction efficiency of α-hydroxy acid, which can be applied to the detection of all α-hydroxy acid introduction.

[0029] A second aspect of the present invention provides a plasmid system, comprising:

[0030] (1) a first expression cassette, wherein the first expression cassette contains a first coding sequence encoding a target protein, wherein the first coding sequence contains a codon for introducing a predetermined α-hydroxy acid, wherein the codon is TAG; and

[0031] (2) a second expression cassette, wherein the second expression cassette contains a second coding sequence encoding a mutant aminoacyl-tRNA synthetase;

[0032] The plasmid system is used to introduce a predetermined α-hydroxy acid into a target protein or to prepare a target protein containing an α-hydroxy acid.

[0033] As described above, the method for introducing α-hydroxy acid into protein of the present invention has the following beneficial effects: the present invention designs a method for effectively introducing α-hydroxy acid into protein by applying a mutant aminoacyl-tRNA synthetase capable of recognizing α-hydroxy acid; the method uses fluorescent protein EGFP as a reporter gene to construct plasmid pBAD-EGFP (149TAG), the 149th amino acid base sequence of the EGFP is mutated to the stop codon TAG, and the EGFP can be used to detect the introduction efficiency of α-hydroxy acid, and can be applicable to the detection of all α-hydroxy acid introduction. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the plasmid map of pEvolve-MmRStRNA disclosed in the present invention.

[0035] Figure 2 This is a comparison chart of the introduction efficiency of different α-hydroxy acids in the examples of the present invention.

[0036] Figure 3 This is the mass spectrometry verification result of EGFP in Example 3 of the present invention. DETAILED DESCRIPTION

[0037] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0038] Example 1

[0039] Construction of recombinant plasmid

[0040] The nucleotide sequence shown in SEQ ID NO.2 was constructed on the vector pEvolve to obtain the pEvolve-MmRStRNA plasmid (plasmid map as shown in Figure 1 ), four rounds of mutation were performed using PCR technology, and the primers used in the process were shown in SEQ ID NO.3 to SEQ ID NO.12.

[0041] (1) The first round of construction included the first round of mutations at sites 346 and 348, using the pEvolve-MmRStRNA plasmid as a template. The mutation primers were as follows:

[0042] N346M / C348W_F: 5'-GCTGATGTTCTGGCAGATGGGTTCTGGTTGCACC-3' (SEQ ID NO.3);

[0043] N346M / C348W_R: 5'-TCTGCCAGAACATCAGCATGGTGAATTCTTCCAGG-3'(SEQ IDNO.4);

[0044] The reaction system is as follows:

[0045]

[0046] (2) Based on the first round of mutation, the second round (Y384F) MmPlyRS point mutation was performed, using the first round of mutation product as a template. The reaction system and the primers consistent with the first round of amplification are shown in SEQ ID NO.5~SEQ ID NO.6:

[0047] Y384F_F: 5'-CATGGTTTTTCGGTGACACCCTGGACGTTATGC-3' (SEQ ID NO.5);

[0048] Y384F_R: 5'-TGTCACCGAAAACCATGCAAGAGTCACCAACG-3' (SEQ ID NO. 6);

[0049] (3) Based on the second round of mutation, point mutations were performed on V401A, V401K, and W417H, respectively. The second round of mutation products were used as templates and amplification was performed with the corresponding primers for each mutation. The reaction system was the same as above. The primer pairs were shown in SEQ ID NO.7 to SEQ ID NO.8, SEQ ID NO.9 to SEQ ID NO.10, and SEQ ID NO.11 to SEQ ID NO.12, respectively:

[0050] V401A_F: 5'-TTCTGCTGCTGTTGGTCCGATCCCGCTGGACC-3' (SEQ ID NO.7);

[0051] V401A_R: 5'-GACCAACAGCAGCAGAAGACAGTTCCAGGTCAC-3' (SEQ ID NO.8);

[0052] V401K_F: 5'-TCTGCTAAAGTTGGTCCGATCCCGCTGGACCG-3' (SEQ ID NO.9);

[0053] V401K_R: 5'-GGACCAACTTTAGCAGAAGACAGTTCCAGGTCAC-3' (SEQ ID NO. 10);

[0054] W417H_F: 5'-ACAAACCGCACATCGGTGCTGGTTTCGGTCTG-3' (SEQ ID NO. 11);

[0055] W417H_R: 5'-ACCGATGTGCGGTTTGTCGATACCCCATTCAC-3' (SEQ ID NO. 12);

[0056] (4) Based on the third-round mutation product N346M / C348W / Y384F / V401A, the fourth round of W417H site mutation was performed, using the mutation product (N346M / C348W / Y384F / V401A) MmPlyRS as a template, and the mutation primers are shown in SEQ ID NO.11 to SEQ ID NO.12.

[0057] The DNA size of the mutant products in each round was verified by agarose gel electrophoresis. The electrophoresis band was located between 5000-7500 bp, which was consistent with the size of pEvolve-MmRStRNA (5600 bp).

[0058] After verifying the bands of the mutation products in each round, take the remaining PCR products and add DpnI enzyme at 38°C for 30 minutes to cut the plasmid template. The reaction system is as follows:

[0059]

[0060] The digested products were transferred into commercial competent cells E.coli DH5α for culture. Single colonies were picked for sequencing verification, and recombinant plasmids pEvolve-MmPylRS(N346M / C348W / Y384F), pEvolve-MmPylRS(N346M / C348W / Y384F / V401A), pEvolve-MmPylRS(N346M / C348W / Y384F / V401K), pEvolve-MmPylRS(N346M / C348W / Y384F / W417H), and pEvolve-MmPylRS(N346M / C348W / Y384F / V401A / W417H) were obtained.

[0061] Example 2

[0062] Construction of reporter protein plasmid

[0063] Using EGFP as the reporter gene, the fluorescent protein sequence is as follows:

[0064] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNR IELKGIDFKEDGNILGHKLEYNYNSH*VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHHHHHH*.

[0065] The site-directed mutagenesis method known to those skilled in the art was used to mutate the 149th amino acid base sequence of EGFP into the stop codon TAG by PCR, which was then introduced into the vector pBAD to construct the reporter protein plasmid pBAD-EGFP (149TAG).

[0066] Example 3

[0067] (1) Synthesis of 2-hydroxyheptanoic acid

[0068] a. Take 1.77 g (77.0 mmol) of Na and dissolve it in 80 mL of ethanol. Add 15.2 g (70.0 mmol) of diethyl acetamidomalonate and stir at room temperature for 40 min. Add 1.162 g (7.0 mmol) of KI and 11.6 g (77.0 mmol) of bromopentane. Heat and reflux at 80° C. for 14 h using a heat-collecting constant temperature heating magnetic stirrer. After the reaction is complete as detected by TLC, the reaction solution is concentrated to a small volume by rotary evaporation. Add 100 mL of distilled water, slowly precipitate solid, filter with suction, and dry naturally to obtain a white solid powder.

[0069] b. Take 6.8g (30.0mmol) of 2-(N-acetylamino)-2-n-pentyl-malonic acid diethyl ester (the above white solid powder) to a 250mL round-bottom flask, add 42mL (84.0mmol) of 2mol / L NaOH, and place the round-bottom flask in a heat-collecting constant temperature heating magnetic stirrer for heating and reflux for 8h; after cooling to room temperature, add 22mL (264mmol) of concentrated hydrochloric acid, and heat and reflux again for 5h. After TLC detection of complete reaction, the reaction solution is concentrated to a small volume by vacuum rotary evaporation, cooled and crystallized, and filtered and dried to obtain solid crystals;

[0070] c. The above solid crystals (4.79 g, 33.0 mmol) were suspended in water and cooled to 2°C. 180 mL of an aqueous solution diluted with sulfuric acid (26.6 mL, 495.5 mmol) and 66 mL of an aqueous solution dissolved with NaNO2 (9.12 g, 132.15 mmol) were added dropwise. The reaction solution was stirred at room temperature overnight, and then extracted with ethyl acetate (30 mL × 3). The organic layers were combined; the organic layer was washed twice with a saturated NaCl solution, the organic layer was separated, anhydrous MgSO4 was added, and the mixture was allowed to stand for 30 min, filtered, and the filtrate was subjected to vacuum rotary evaporation to remove the solvent to obtain an oily product, i.e., 2-hydroxyheptanoic acid.

[0071] (2) Introduction of 2-hydroxyheptanoic acid

[0072] The recombinant plasmid pEvolve-MmPylRS (N346M / C348W / Y384F) prepared in Example 1 and the reporter protein plasmid pBAD-EGFP (149TAG) were transformed into the host cell E. coli DH10B. A single colony was obtained on the resistance plate and induced. The specific operation was as follows:

[0073] Pick a healthy single colony from the solid plate and inoculate it into 3 mL of LB liquid culture medium containing ampicillin (100 μg / mL) and chloramphenicol (100 μg / mL) resistance, and culture it in a 37°C constant temperature oscillator overnight. The next day, add the bacterial liquid to 3 mL of LB liquid culture medium containing ampicillin (100 μg / mL) and chloramphenicol (100 μg / mL) at a ratio of 1 (bacterial liquid): 50 (culture medium) and culture it until the OD 600 =0.6~0.8, then add 15% concentration of arabinose and 2-hydroxyheptanoic acid at a volume ratio of 1:100 (bacterial solution), and continue to culture overnight in a constant temperature oscillator at 37℃. Take 1.5mL of the induced bacterial solution and centrifuge at 12000rpm for 1min, discard the culture medium, add 1mLddH2O to suspend and wash, centrifuge to remove the supernatant, repeat twice, and finally add 1.5mLddH2O to suspend the bacteria.

[0074] Example 4

[0075] (1) Synthesis of S-allylcysteine

[0076] a. Take 6 g of cysteine ​​and dissolve it in 28% NH3ꞏH2O (16 mL), add 13.5 g of allyl bromide (52.0 mmol) dropwise into the reaction solution, precool for 15 min and stir for 12 h; the reaction solution is rotary evaporated at 40 °C to remove the solvent, and the residual allyl bromide is washed with ethanol to obtain solid crystals;

[0077] b. The above solid crystals (5.31 g, 33.0 mmol) were suspended in water and cooled to 2°C. 180 mL of an aqueous solution diluted with sulfuric acid (26.6 mL, 495.5 mmol) and 66 mL of an aqueous solution dissolved with NaNO2 (9.12 g, 132.15 mmol) were added dropwise. The reaction solution was stirred at room temperature overnight, and then extracted with ethyl acetate (30 mL × 3). The organic layers were combined, washed twice with a saturated NaCl solution, separated, anhydrous MgSO4 was added, and the mixture was allowed to stand for 30 min. The mixture was filtered, and the filtrate was subjected to vacuum rotary evaporation to remove the solvent to obtain an oily product, i.e., S-allylcysteine.

[0078] (2) Introduction of S-allylcysteine

[0079] The recombinant plasmid pEvolve-MmPylRS (N346M / C348W / Y384F) prepared in Example 1 and the reporter protein plasmid pBAD-EGFP (149TAG) were transformed into the host cell E. coli DH10B. A single colony was obtained on the resistance plate and induced. The specific operation was as follows:

[0080] Pick a healthy single colony from the solid plate and inoculate it into 3 mL of LB liquid culture medium containing ampicillin (100 μg / mL) and chloramphenicol (100 μg / mL) resistance, and culture it in a 37°C constant temperature oscillator overnight. The next day, add the bacterial liquid to 3 mL of LB liquid culture medium containing ampicillin (100 μg / mL) and chloramphenicol (100 μg / mL) at a ratio of 1 (bacterial liquid): 50 (culture medium) and culture it until the OD 600 =0.6~0.8, then add 15% concentration of arabinose and S-allyl cysteine ​​at a volume ratio of 1:100 (bacterial solution), and continue to culture overnight in a constant temperature oscillator at 37℃. Take 1.5mL of the induced bacterial solution and centrifuge at 12000rpm for 1min, discard the culture medium, add 1mL ddH2O to suspend and wash, centrifuge to remove the supernatant, repeat twice, and finally add 1.5mL ddH2O to suspend the bacteria.

[0081] Example 5

[0082] (1) Synthesis of S-tert-butyl cysteine

[0083] a. Dissolve 12.1 g of cysteine ​​in a mixed solution of 45 mL of hydrochloric acid and 12.5 mL (130 mmol) of tert-butanol, reflux at room temperature for 10 h, evaporate the reaction solution by rotary evaporation, filter, and wash with acetone to obtain solid crystals;

[0084] b. Take the above solid crystals (5.84 g, 33.0 mmol) and suspend them in water and cool them to 2°C. Add 180 mL of aqueous solution diluted with sulfuric acid (26.6 mL, 495.5 mmol) and 66 mL of aqueous solution dissolved with NaNO2 (9.12 g, 132.15 mmol) dropwise. Stir the reaction solution at room temperature overnight, extract with ethyl acetate (30 mL × 3), combine the organic layers, wash the organic layers with saturated NaCl solution twice, separate the organic layers, add anhydrous MgSO4, let stand for 30 min, filter, and remove the solvent by vacuum rotary evaporation to obtain an oily product, i.e., S-tert-butyl cysteine.

[0085] (2) Introduction of S-tert-butyl cysteine

[0086] The recombinant plasmid pEvolve-MmPylRS (N346M / C348W / Y384F) prepared in Example 1 and the reporter protein plasmid pBAD-EGFP (149TAG) were transformed into the host cell E. coli DH10B. A single colony was obtained on the resistance plate and induced. The specific operation was as follows:

[0087] Pick a healthy single colony from the solid plate and inoculate it into 3 mL of LB liquid culture medium containing ampicillin (100 μg / mL) and chloramphenicol (100 μg / mL) resistance, and culture it in a 37°C constant temperature oscillator overnight. The next day, add the bacterial liquid to 3 mL of LB liquid culture medium containing ampicillin (100 μg / mL) and chloramphenicol (100 μg / mL) at a ratio of 1 (bacterial liquid): 50 (culture medium) and culture it until the OD 600 =0.6~0.8, then add 15% arabinose and S-tert-butyl cysteine ​​at a ratio of 1:100 (bacterial solution), and continue to culture overnight in a constant temperature oscillator at 37°C. Take 1.5mL of the induced bacterial solution and centrifuge at 12000rpm for 1min, discard the culture medium, add 1mL ddH2O to suspend and wash, centrifuge to remove the supernatant, repeat twice, and finally add 1.5mL ddH2O to suspend the bacteria.

[0088] Example 6

[0089] Validation of the introduction of α-hydroxy acids

[0090] Take 1.5 mL of the induced bacterial solution in Examples 3 to 5 and test its OD 600 The fluorescence intensity of the induced bacterial solution was compared to determine the introduction efficiency of 2-hydroxyheptanoic acid, S-allylcysteic acid, and S-tert-butylcysteic acid; the control group was an induced bacterial solution induced under the same conditions without adding α-hydroxy acid. The test results are shown in Table 1 and Figure 2 shown.

[0091] Table 1. Introduction efficiency of different α-hydroxy acids

[0092]

[0093] According to the test results, when α-hydroxy acid is not introduced, EGFP cannot express a complete protein with fluorescent properties, while EGFP that can recognize and introduce α-hydroxy acid at position 149 expresses a complete fluorescent protein. Using the enhanced green fluorescent protein gene (EGFP) as a reporter gene to detect the introduction efficiency of α-hydroxy acid can be applied to the detection of all α-hydroxy acid introduction. Among them, EGFP has the highest introduction efficiency for 2-hydroxyheptanoic acid.

[0094] Example 7

[0095] Mass spectrometry verification

[0096] The mutated EGFP amino acid sequence was calculated on the protein molecular weight software and its molecular weight X should be equal to (the molecular weight of the successfully expressed protein) + (the molecular weight of α-hydroxy acid) - 18.0 (dehydrated peptide bond) - 18.0 (GFP chromophore - 18 dehydrate).

[0097] On the one hand, according to the EGFP introduced with 2-hydroxyheptanoic acid in Example 3, X=27650.14 (molecular weight of successfully expressed protein)+146.19 (molecular weight of 2-hydroxyheptanoic acid)-18.0 (dehydrated peptide bond)-18.0 ((GFP chromophore-18 dehydrated))=27760 was calculated.

[0098] On the other hand, the mass spectrometry data is used to verify whether the introduction is successful. Figure 3 , X=27759, machine calibration offset 1 (standard), molecular weight is correct, indicating that 2-hydroxyheptanoic acid was successfully introduced.

[0099] In summary, the present invention obtains a method for effectively introducing α-hydroxy acid into protein by using a mutant aminoacyl tRNA synthetase capable of recognizing α-hydroxy acid; the method uses fluorescent protein EGFP as a reporter gene, the 149th amino acid base sequence of the EGFP is mutated to the stop codon TAG, and the EGFP can be used to detect the introduction efficiency of α-hydroxy acid, and can be applied to the detection of all α-hydroxy acid introduction. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

[0100] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for introducing α-hydroxy acid into protein, characterized in that: The following steps are involved: (1) Construction of recombinant plasmid and target protein plasmid; The recombinant plasmid contains a gene encoding a mutant aminoacyl-tRNA synthetase, wherein the mutant aminoacyl-tRNA synthetase is mutated corresponding to the aminoacyl-tRNA synthetase of the wild-type Methanosarcina mazei whose amino acid sequence is shown in SEQ ID NO.1, and the mutation is as follows: asparagine (N) at position 346 is mutated to methionine (M), cysteine ​​(C) at position 348 is mutated to tryptophan (W), and tyrosine (Y) at position 384 is mutated to phenylalanine (F); The target protein plasmid contains a gene encoding the target protein, and the site where the α-hydroxy acid is to be introduced into the target protein plasmid is mutated to a stop codon TAG; (2) The recombinant plasmid and the target protein plasmid are co-transformed into host cells, and the host cells are induced and cultured while adding α-hydroxy acid.

2. The method for introducing α-hydroxy acid into protein according to claim 1, characterized in that: The α-hydroxy acid is a compound having both a hydroxyl group and a carboxyl group on the α-carbon, and its structure is shown in formula (I): (I)； Wherein, R can be any group, including but not limited to the side chain of natural amino acids.

3. The method for introducing α-hydroxy acid into protein according to claim 2, characterized in that: The α-hydroxy acid includes any one of 2-hydroxyheptanoic acid, S-allylcysteic acid, and S-tert-butylcysteic acid.

4. The method for introducing α-hydroxy acid into protein according to claim 1, characterized in that: The recombinant plasmid is pEvolve-MmRStRNA; the target protein is any protein that needs to introduce α-hydroxy acid.

5. The method for introducing α-hydroxy acid into protein according to claim 1, characterized in that: The recombinant plasmid / target protein plasmid also contains one or more of a resistance gene, a tag sequence, a repressor protein gene, and a promoter gene.

6. The method for introducing α-hydroxy acid into protein according to claim 5, characterized in that: The resistance gene is selected from one or more of the group consisting of ampicillin resistance gene, chloramphenicol resistance gene, kanamycin resistance gene and tetracycline resistance gene.

7. The method for introducing α-hydroxy acid into protein according to claim 1, characterized in that: The host cell includes Escherichia coli; when the Escherichia coli is cultured, the culture temperature is 37°C, the inducer includes arabinose, and the concentration of α-hydroxy acid is 1%.

8. The method for introducing α-hydroxy acid into protein according to claim 1, characterized in that: EGFP was used as a reporter protein, the 149th amino acid base sequence of the EGFP was mutated to a termination codon TAG, and a reporter protein plasmid pBAD-EGFP (149TAG) was constructed. The reporter protein plasmid contained a gene encoding the reporter protein.

9. A plasmid system, characterized in that The method for introducing α-hydroxy acid into protein as claimed in any one of claims 1 to 8 comprises: (1) a first expression cassette, wherein the first expression cassette contains a first coding sequence encoding a target protein, wherein the first coding sequence contains a codon for introducing a predetermined α-hydroxy acid, wherein the codon is TAG; and (2) a second expression cassette, wherein the second expression cassette contains a second coding sequence encoding a mutant aminoacyl-tRNA synthetase; The plasmid system is used to introduce a predetermined α-hydroxy acid into a target protein or to prepare a target protein containing an α-hydroxy acid.

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