Seryl tRNA synthetase mutants and their applications

Abstract

This invention discloses a serine tRNA synthetase mutant and its applications, belonging to the field of enzyme proteins. This invention utilizes machine learning techniques such as AlphaFold3 to predict SerRS and tRNA... Sec The interaction interface, thereby precisely locating the SerRS that affects tRNA Sec By identifying key amino acid residues and employing a semi-rational mutation strategy, SerRS key sites were targeted and modified to enhance their binding to tRNA. Sec By combining binding affinity and catalytic efficiency, a high tRNA binding affinity and catalytic efficiency were constructed. Sec The SerRS variant with aminoacylation activity improves the supply level of Sec, enabling efficient and high-fidelity expression of selenoproteins, eliminating the mis-incorporation of Ser, and providing a new technical means for the biosynthesis of selenoproteins.

Description

Technical Field

[0001] This invention belongs to the field of enzyme proteins, specifically relating to a serine tRNA synthetase mutant and its applications. Background Technology

[0002] Selenium is an essential trace element for the human body, mainly existing in the form of selenocysteine ​​(Sec), participating in various physiological processes, including antioxidation, signal transduction, and immune regulation. The active site of selenoproteins is usually composed of Sec, which has a stronger ability to resist oxidative inactivation compared to cysteine ​​(Cys). Therefore, selenoproteins play an important role in preventing major diseases such as cardiovascular disease, diabetes, and cancer. However, common inorganic selenium products are difficult for the human body to absorb and utilize, and there are almost no commercially available selenoprotein products. Heterologous protein expression using microbial cell factories is a common protein production method. Some bacteria naturally possess Sec synthesis and incorporation systems, such as *Escherichia coli*, commonly used in amino acid fermentation and protein expression, which has been used to express bacterial and human selenoproteins. In these bacteria, Sec synthesis uses serine (Ser) as a precursor; however, unlike other amino acids, Sec synthesis must be carried out in an aminoacylated form. The first step is loading Ser into the tRNA of Sec (tRNA). Sec The reaction is catalyzed by serine synthase (SerRS), which simultaneously recognizes Ser's tRNA (tRNA). Ser ) and tRNA Sec Ser was loaded onto two types of tRNA, generating their respective aminoacyl-tRNAs. The former is responsible for introducing Ser into the peptide chain during translation, while the latter, under the action of an enzyme, converts the serine hydroxyl group to a selenoyl group, generating an aminoacylated Sec-tRNA. Sec This is then used for the incorporation of Sec. Due to the structural differences in tRNA, SerRS, which is responsible for aminoacylation, is more likely to recognize Ser tRNA, while it is more likely to recognize tRNA. Sec The aminoacylation efficiency is only that of tRNA Ser 1%. Despite overexpression of tRNA Sec This can alleviate the bottleneck, but it can only increase the final Sec synthesis level by about 10%, mainly because SerRS and tRNA... Sec The affinity for tRNA is low. In contrast, the catalytic enzymes in subsequent pathways have a lower affinity for tRNA. Sec The matching degree is extremely high, and it has the ability to convert Ser-tRNA to concentrations exceeding physiological levels. Sec Therefore, tRNA Sec Aminoacylation has become the rate-limiting step in the synthesis of Sec and selenoproteins. Semi-rational design was used to modify SerRS to improve its response to tRNA. SecIts affinity can significantly improve the synthesis efficiency of selenoproteins, thus providing a more effective strategy for solving the problem of selenium deficiency.

[0003] MUKAI T, SEVOSTYANOVAA, SUZUKI T, et al. Afacile method for producing selenocysteine-containing proteins[J]. Angew Chem Int Ed Engl, 2018, 57(24):7215-9. doi:10.1002 / anie.201713215 discloses methods for producing selenocysteine-containing proteins using tRNA. Sec tRNA Ser Based on allo-tRNAs with long receptor arms and short T arms, artificial tRNAs such as SecUX, which can be recognized by the universal elongation factor EF-Tu, were obtained by replacing and fine-tuning the domains that bind to elongation factors in these tRNAs. This allows Sec-loaded artificial tRNAs to autonomously decode like other types of tRNAs, achieving Sec incorporation at any site. However, in this study, the EF-Tu elongation factor resulted in significant mis-incorporation of Ser, leading to low incorporation rates and low purity of the synthesized selenoprotein. Furthermore, these artificial tRNAs still could not bind efficiently to SerRS, and the expression level of selenoprotein remained dependent on aminoacyl-tRNA. Sec Limited supply.

[0004] FU X, CRNKOVIC A, SEVOSTYANOVAA, et al. Designing seryl-tRNA synthetase for improved serylation of selenocysteine ​​tRNAs[J]. FEBS Lett, 2018, 592(22):3759-68. doi:10.1002 / 1873-3468.13271 discloses the evolution of three ser-tRNAs that improve serylation of selenocysteine ​​tRNAs under the guidance of structural modeling and chloramphenicol acetyltransferase activity screening. Sec The synthesized SerRS variants are as follows:

[0005] 1.K86C:A87Y:D90F:S160T:D228K:T229M;

[0006] 2.K86K:A87T:D90V:S160N:D228R:T229N;

[0007] 3.K86A:A87L:D90T:S160P:D228R:T229N;

[0008] For example, K86C means that the lysine at position 86 of the SerRS enzyme protein's amino acid sequence is mutated to cysteine. Compared to wild-type SerRS, these three SerRS mutants showed a 10-fold, 8-fold, and 4-fold increase in kJ / mL, respectively, when using artificially modified tRNA as a substrate. cat / K M However, this literature focuses on the enzymatic modification of engineered tRNA, and does not address the problem of Ser misincorporation caused by the use of artificial tRNA. Furthermore, it only demonstrates that the SerRS variant has an increased catalytic constant for aminoacylation of artificial tRNA, without actually proving an increase in selenoprotein expression. Sec synthesis and selenoprotein expression remain limited. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a SerRS mutant enzyme with higher catalytic efficiency, which can effectively improve the expression level, enzyme activity and purity of selenoprotein.

[0010] The technical solution of this invention is: a serine tRNA synthetase mutant, which is obtained by mutation of any one of a to d or by any combination of d with a, b or c based on the serine tRNA synthetase shown in SEQ ID NO.1.

[0011] a) The amino acid at position 229 is mutated from threonine to arginine;

[0012] b. The 300th amino acid is mutated from aspartic acid to valine;

[0013] c. The 363rd amino acid was mutated from tryptophan to phenylalanine;

[0014] d. The 382nd amino acid is mutated from threonine to alanine, valine, or serine.

[0015] A gene encoding the serine tRNA synthetase mutant described above.

[0016] Expression vectors containing the genes described above.

[0017] Engineered bacteria containing the expression vector described above.

[0018] The application of the mutants, genes, expression vectors, or engineered bacteria described above in the synthesis of selenoproteins.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] To improve the synthesis and incorporation efficiency of sec and increase selenoprotein synthesis, this invention provides a SerRS mutant enzyme with higher catalytic efficiency, which can effectively improve selenoprotein expression, enzyme activity, and purity. The biosynthesis of selenoproteins depends on the efficient incorporation of selenocysteine ​​(Sec), and the synthesis of sec and tRNA loading are limited by the catalytic efficiency of SerRS. The amylation reaction catalyzed by SerRS is the key rate-limiting step. *E. coli* SerRS enhances the tRNA... Sec The aminoacylation efficiency is only that of tRNA Ser 1%, because of tRNA Sec With tRNA Ser Significant differences exist in secondary structure and sequence, leading to SerRS's dependence on tRNA. Sec The recognition and binding ability of SerRS is relatively weak. This invention utilizes machine learning techniques such as AlphaFold3 to predict SerRS and tRNA. Sec The interaction interface, thereby precisely locating the SerRS that affects tRNA Sec By identifying key amino acid residues and employing a semi-rational mutation strategy, SerRS key sites were targeted and modified to enhance their binding to tRNA. Sec By combining binding affinity and catalytic efficiency, a high tRNA binding affinity and catalytic efficiency were constructed. Sec The SerRS variant with aminoacylation activity improves the supply level of Sec, enabling efficient and high-fidelity expression of selenoproteins, eliminating the mis-incorporation of Ser, and providing a new technical means for the biosynthesis of selenoproteins. Attached Figure Description

[0021] Figure 1 Alphafold 3 simulations predict, where (a) Alphafold 3 predicts SerRS-tRNA. Ser (b) Alphafold 3 predicts SerRS-tRNA Sec Composite structure.

[0022] Figure 2 The growth of strains in an environment with added carbenicillin antibiotics indicates changes in the catalytic effect of SerRS, including (a) the effect of replacing Cys70 and Cys240 residues of β-lactamase with other residues on the ability of strains to resist carbenicillin; (b) the cell density of strains under carbenicillin stress when the 70th Cys codon of β-lactamase was replaced with the UGA codon encoding Sec and introduced into strains containing different selenoprotein synthesis elements, and the control strain did not contain selenoprotein synthesis elements.

[0023] Figure 3The growth of numbered bacteria in the single-strain screening of the SerRS_T382 mutant library under screening pressure was observed. Mutant libraries containing different mutation directions of the T382 site of SerRS were screened. Under the same screening conditions, compared with strains expressing wild-type SerRS, strains expressing most T382 variants showed significantly faster growth rates, indicating that the catalytic efficiency of the T382 site variants of SerRS was significantly improved.

[0024] Figure 4 The growth of numbered bacteria in the single-strain screening of the SerRS_W363 mutant library under screening pressure was observed. Mutant libraries containing different mutation directions at the W363 site of SerRS were screened. Under the same screening conditions, strains expressing most W363 variants showed significantly faster growth rates compared to strains expressing wild-type SerRS, indicating that the catalytic efficiency of the W363 site variants of SerRS was significantly improved.

[0025] Figure 5 Growth of SerRS variant T382A and SerRS wild-type overexpressing strains under selection pressure; Under the same selection conditions, compared with the strain expressing wild-type SerRS, the strain expressing SerRS variant T382A grew significantly faster, indicating that the catalytic efficiency of SerRS variant T382A was significantly improved.

[0026] Figure 6 The growth of SerRS mutant T229R, SerRS wild-type overexpressing strains, and blank control strains under selection pressure; under the same selection conditions, compared with strains expressing wild-type SerRS, strains expressing SerRS variant T229R grew significantly faster, indicating that SerRS variant T229R has significantly improved catalytic efficiency.

[0027] Figure 7 The growth of SerRS mutant D300V, SerRS wild-type overexpressing strains, and blank control strains under selection pressure; under the same selection conditions, compared with strains expressing wild-type SerRS, strains expressing SerRS variant D300V grew significantly faster, indicating that SerRS variant D300V has significantly improved catalytic efficiency.

[0028] Figure 8The growth of SerRS variant D300V:T382A, SerRS wild-type overexpressing strains, and blank control strains under selection pressure, and the gene sequencing results of mutation sites; under the same selection conditions, compared with strains expressing wild-type SerRS, strains expressing SerRS variant D300V:T382A showed significantly faster growth rates, indicating that SerRS variant D300V:T382A significantly improved catalytic efficiency. Gene sequencing results showed that the aspartic acid (Asp) at position 300 of SerRS was mutated to valine (Val), and the threonine (Thr) at position 382 was mutated to alanine (Ala).

[0029] Figure 9 Growth of SerRS variant W363F:T382V and SerRS wild-type overexpressing strains under selection pressure; Under the same selection conditions, compared with strains expressing wild-type SerRS, strains expressing SerRS variant W363F:T382V grew significantly faster, indicating that SerRS variant W363F:T382V had significantly improved catalytic efficiency.

[0030] Figure 10 The growth of SerRS variant T229R:T382S and SerRS wild-type overexpressing strains under different selection pressures is shown in the figure. Different concentrations of Carb and Serine were set. Under the conditions of adding 45 mg / L Carb and 1 g / L Ser, the growth rate of the strain expressing SerRS variant T229R:T382S was significantly faster than that of the strain expressing wild-type SerRS, indicating that the catalytic efficiency of SerRS variant T229R:T382S was significantly improved.

[0031] Figure 11 Using the SerRS mutant T382A from this invention to express mouse selenoprotein-glutathione peroxidase significantly increases the expression levels of total protein and full-length selenoprotein compared to overexpressing wild-type SerRS alone.

[0032] Figure 12 Mass spectrometry identification results of mouse selenoprotein-glutathione peroxidase expressed using the SerRS mutant T382A of this invention.

[0033] Figure 13 Using the SerRS mutant T382A of this invention can significantly increase the expression level of selenoformate dehydrogenase FdhH. Detailed Implementation

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0035] Example 1 Experimental Method

[0036] The biosynthesis of selenoproteins depends on the efficient incorporation of selenocysteine ​​(Sec), and the synthesis of Sec and tRNA loading are limited by the catalytic efficiency of SerRS. The SerRS-catalyzed aminoacylation reaction is the key rate-limiting step. *E. coli* SerRS-catalyzed tRNA... Sec The aminoacylation efficiency of tRNA is only 1% of that of tRNASer, because tRNA Sec With tRNA Ser Significant differences exist in secondary structure and sequence, leading to SerRS's dependence on tRNA. Sec The recognition and binding ability of SerRS is relatively weak. This invention utilizes machine learning techniques such as AlphaFold3 to predict SerRS and tRNA. Sec The interaction interface, thereby precisely locating the SerRS that affects tRNA Sec By identifying key amino acid residues and employing a semi-rational mutation strategy, SerRS key sites were targeted and modified to enhance their binding to tRNA. Sec By combining binding affinity and catalytic efficiency, a high tRNA binding affinity and catalytic efficiency were constructed. Sec A SerRS variant with aminoacylation activity. The specific steps are as follows:

[0037] (1) tRNA Sec - Three-dimensional model establishment of the SerRS complex:

[0038] This approach establishes a rationally designed mutant library using two prediction methods: a classical molecular docking prediction method and an Alphafold 3 prediction method. The modeling method utilizes the amino acid sequence of the SerRS protein and the base sequence of tRNA to establish a three-dimensional interaction model for analyzing the interaction sites between SerRS and tRNA. The SerRS used in this invention is the basal serS gene expression of *Escherichia coli* strain MG1655.K12, with a Gene ID of 945506 in GenBank.

[0039] Classical molecular docking simulation prediction: based on the tRNA of E. coli MG1655 Sec Base sequence and two-dimensional structure (RNAcentral ID: URS00002F9CDD_511145), tRNA prediction using simRNA2.0 tool. Sec The three-dimensional structure of tRNA was obtained (https: / / genesilico.pl / SimRNAweb), with the calculation parameters set to the website's default settings. tRNA was then constructed. SecAfter obtaining the 3D structure, the SerRS 3D structure of *E. coli* (PDB ID: 6R1M) was downloaded. Water molecules and small ligands in the SerRS molecular structure were removed using Discovery Studio 4.5. Autodock Vina was used to predict the docking of the two molecules, with docking parameters: center x = -6.965, center y = 1.972, center z = -0.051; size x = 126.0, size y = 126.0, size z = 126.0, energy range = 4, exhaustiveness = 12, and num modes = 10. Finally, further analysis was performed based on the docking model with the highest score. Figure 1 ).

[0040] Alphafold 3 simulation prediction: based on the tRNA of E. coli MG1655 Sec The base sequence (RNAcentral ID: URS00002F9CDD_511145) and the protein sequence of *E. coli* SerRS (PDB ID: 6R1M) were used to predict two tRNAs using Alphafold3. Sec The complex structure with the SerRS protein dimer is shown in the image (https: / / golgi.sandbox.google.com / ). Three molecules were added for docking, including two RNA molecules, both with tRNA sequences. Sec The system inputs two SerRS protein sequences as the dimer protein sequences, using the base sequence and a SerRS protein dimer. The prediction parameters are all the website's default parameters. Finally, further analysis is performed based on the highest-scoring prediction model (default is model_0).

[0041] (2) Establishment of the mutation library:

[0042] Interaction region analysis and establishment of a rational mutation library: for the obtained tRNA Sec The three-dimensional structure of the SerRS complex was obtained, and Pymol 3.0 was used to screen for SerRS molecules that interact with tRNA. Sec Amino acid residue sites with intermolecular forces were used to screen SerRS molecules for interactions with tRNA. Sec For amino acid residue sites with intermolecular distances within 4 angstroms, various mutant libraries were established for these sites, which have the potential to enhance the catalytic activity of SerRS according to theoretical analysis.

[0043] Semi-rational mutation libraries: Amino acid residue sites obtained through two screening methods were used as a union to construct single-site saturated mutation libraries (the original amino acid sites mutated in the directions of the 20 classical amino acids). Simultaneously, for screening sites less than 150 bp apart in the gene sequence, multi-site combined saturated mutation libraries (multiple sites simultaneously containing the mutation directions of the 20 classical amino acids) and multi-site combined random primer mutation libraries were constructed (the original base ratio of the amino acid site was 85%, and the remaining three bases each accounted for 5%, controlling the mutation rate and protecting the normal function of the protein).

[0044] Establishment of a random mutation library: For the entire SerRS gene, error-prone PCR was performed on the entire SerRS gene using primers 5'-gtatttatgacggacgattgtggc-3' and 5'-cggcggatttgtcctactca-3' to establish an error-prone mutation library (3‰-6‰ of the bases in the entire SerRS gene are randomly mutated).

[0045] (3) Construction of the screening system

[0046] This scheme employs a carbenicillin-based screening method, using the growth of strains in an environment containing carbenicillin to indicate changes in SerRS catalytic activity. β-lactamases can cleave carbenicillin antibiotics; strains correctly expressing this enzyme can grow in an environment containing carbenicillin, while those not expressing it cannot. Some β-lactamases contain Cys residues that can form disulfide bonds, which are crucial for maintaining enzyme activity. Theoretically, these Cys residues can be replaced with similarly morphologically similar Sec residues to form selenium-sulfur bonds or diselenoses. However, mutations to other amino acid residues will disrupt the enzyme structure and fail to confer resistance to carbenicillin antibiotics. Figure 2 In other words, the gene can specifically distinguish Sec from other amino acids, thus it can be used to specifically indicate the level of Sec synthesis and incorporation.

[0047] Starting from the 5' end, the Cys codons in the β-lactamase gene were sequentially replaced with UAG to construct expression plasmids. β-lactamase expression plasmids containing different UAG frequencies were then compared with those carrying only tRNA. Sec CUA The expression plasmids were co-transformed into the C321.ΔAΔselACDΔfdhFΔbla strain, with a strain overexpressing wild-type β-lactamase as a control. The growth levels of the strains were measured in LB medium containing an appropriate concentration of carbenicillin to determine the frequency of codon substitutions that could significantly reduce cell concentration. Based on this, 0.5, 1.0, and 2.0 g / L of L-serine were added to the medium to create Ser-tRNA. Sec CUAThe concentration gradient was used to detect bacterial cell growth levels. The phenomenon that bacterial cell concentration increases with the addition of L-serine can be used to indicate tRNA. Sec Increased level of aminoacylation ( Figure 2 (b)

[0048] (4) Iterative screening:

[0049] The SerRS mutant library, wild-type SerRS, and empty plasmid were respectively transformed into the C321.ΔAAT strain (a strain in which the carbenicillin resistance gene frames bla and tetR of the C321.ΔA strain were knocked out) and used as the experimental group, positive control, and negative control, respectively. The SerRS variant with improved catalytic efficacy can confer resistance to higher concentrations of carbenicillin antibiotics to the strain.

[0050] First, a preliminary screening was conducted under a low concentration of carbenicillin antibiotic to identify effective SerRS mutants. For SerRS mutant library cultures that could grow normally under the preliminary screening conditions, the cultures were diluted and plated to obtain single colonies (the cultures may contain multiple SerRS mutants, but each single colony contains only one mutant). Each single colony was then inoculated into a higher concentration of carbenicillin antibiotic for secondary screening under more stringent conditions to identify SerRS mutants with significant advantages.

[0051] After screening, the SerRS mutants with significant advantages in the secondary screening were validated. SerRS variants with the same mutation site were artificially constructed and overexpressed in strains. If, under the selection conditions, strains overexpressing this SerRS variant grew faster than strains overexpressing wild-type SerRS, it indicates that the mutation site of this dominant variant can affect the interaction between SerRS and tRNA. Sec Affinity between them catalyzes tRNA Sec The rate of aminoacylation is increased.

[0052] (5) Synthesis of selenoproteins:

[0053] Formate dehydrogenase (FdhH) and glutathione peroxidase (GPx) are important selenoproteins, both with Sec as their catalytic active site. FdhH or GPx was heterologously expressed in strains overexpressing the SerRS variant. The proteins were extracted and purified, and SDS-PAGE was used to verify protein expression levels. Proteomic analysis was used to confirm the correct incorporation of Sec to verify the purity of the synthesized selenoproteins. Simultaneously, enzyme activity was measured to verify the activity of the expressed proteins.

[0054] Through the above screening, the following mutants of SerRS were obtained: T229R, D300V, W363F, T382A, T382V, and T382S.

[0055] Example 2 verifies the advantages of the SerRS variant of the present invention compared to wild-type SerRS.

[0056] Validation experiments typically involve two or three groups: one expressing wild-type (WT) SerRS and the other expressing a SerRS variant, such as SerRS:T229R, which indicates that the mutation site of this variant is threonine (T) at position 229, and the mutation direction is to arginine (R). The sel-control group represents a blank control group that does not express SerRS. The selection pressure in the validation experiment is mainly regulated by the concentration of the antibiotic carbenicillin sodium (Carb) and the replenishment concentration of serine (Ser). The higher the catalytic efficiency of the SerRS variant, the higher the efficiency of Sec synthesis and incorporation, and the more selenoprotein β-lactamase the bacteria synthesize. This selenoprotein can cleave carbenicillin, allowing the strain to survive under the selection pressure of carbenicillin.

[0057] Carb concentrations are typically set between 30 mg / L and 60 mg / L. Within this range, higher Carb concentrations place greater pressure on the SerRS catalytic efficiency. Ser concentrations are typically set between 0.1 g / L and 4 g / L. Within this range, Ser, as one of the precursor reactants in the SerRS catalytic reaction, releases more SerRS function and facilitates the SerRS catalytic reaction with higher replenishment concentrations. OD 600 OD (Optical Density) refers to the optical density value measured when the wavelength of a spectrophotometer is set to 600 nm. It is one of the commonly used methods for determining bacterial cell count and can indirectly reflect the bacterial concentration in liquid culture media. 600 The higher the concentration of SerRS, the more cells are present in the bacterial culture, and vice versa. Under certain concentrations of Carb and Ser, the growth of *E. coli* carrying the SerRS expression plasmid is used to indicate the catalytic efficiency of SerRS. At the same time, faster cell growth or higher cell density indicates higher catalytic efficiency of the expressed SerRS. Based on the SerRS-tRNA mimicry structure, potentially interacting amino acid sites were selected, and these sites were saturated with mutations to create a single-point saturated mutant library containing 20 different amino acid mutation directions at these sites. The mutant library was expressed in *E. coli*, and initial screening was performed in a medium containing 30 mg / L Carb. Compared to strains expressing only wild-type SerRS, strains expressing SerRS mutant libraries such as T382 grew faster, indicating that the T382 mutation of SerRS... Figure 3 W363 Figure 4 T229 Figure 6 ) and D300 ( Figure 7 Some variants of the Sec site can improve the efficiency of Sec synthesis and incorporation.

[0058] SerRS variants that were initially screened to enhance Sec synthesis and incorporation were expressed in *E. coli*. The effects were validated in a medium containing 60 mg / L Calb. Compared to strains expressing wild-type SerRS, strains expressing SerRS variants such as T382A, T229R, and D300V grew faster, indicating that SerRS variants such as T382A, T229R, and D300V can improve Sec synthesis and incorporation efficiency. Figure 5 , Figure 6 , Figure 7 ).

[0059] Example 3: Validation of the effect of SerRS combined mutation

[0060] The efficacy of expressing SerRS combinatorial mutants in *E. coli* was validated in a medium containing 60 mg / L Carb. Compared with strains expressing wild-type SerRS, strains expressing SerRS combinatorial variants T382A:D300V, W363F:T382V, and T229R:T382S grew faster, indicating that variants such as SerRS:T382A:D300V, W363F:T382V, and T229R:T382S can improve the efficiency of Sec synthesis and incorporation. Figure 8 , Figure 9 , Figure 10 ).

[0061] Example 4: The SerRS variant can increase selenoprotein expression.

[0062] Using the SerRS mutant T382A obtained in this invention, along with other necessary elements for Sec incorporation, a murine selenoprotein—glutathione peroxidase—was expressed and purified in *E. coli* MG1655. SDS-PAGE analysis showed that, compared to overexpression of wild-type SerRS, the SerRS mutant of this invention significantly increased the expression level of selenoprotein. Figure 11 , Figure 12 ).

[0063] Example 5: The SerRS variant can enhance the enzymatic activity and purity of selenoproteins.

[0064] Using the SerRS mutant T382A obtained in this invention, along with other necessary elements for Sec incorporation, expression and purification of mouse selenoprotein-glutathione peroxidase in Escherichia coli MG1655 significantly enhanced the enzyme activity of selenoprotein. DTNB assay showed that, compared to overexpression of wild-type SerRS, this invention increased both protein yield and enzyme activity of selenoprotein (Table 1).

[0065] Table 1 shows that using the SerRS mutant of this invention can improve the enzyme activity and purity of selenoproteins.

[0066] SerRS category Protein yield (mg / L) Enzyme activity (U / mg) The SerRS mutant T382A of this invention 11.99 0.935 Wild-type SerRS 1.40 0.148

[0067] Example 6: The SerRS mutant can increase the expression level of formate dehydrogenase FdhH.

[0068] Using the SerRS mutant T382A obtained in this invention, along with other necessary elements for Sec incorporation, the expression and purification of selenoformate dehydrogenase FdhH in Escherichia coli MG1655 can significantly increase protein expression levels. Figure 13 ).

Claims

1. A serine tRNA synthetase mutant, characterized in that, It is based on the serine tRNA synthetase shown in SEQ ID NO.1, where the amino acid at position 229 is mutated from threonine to arginine, and the amino acid at position 382 is mutated from threonine to serine.

2. A gene encoding a serine tRNA synthetase mutant as described in claim 1.

3. An expression vector containing the gene of claim 2.

4. Engineered bacteria containing the expression vector of claim 3.

5. The application of the mutant of claim 1, the gene of claim 2, the expression vector of claim 3, or the engineered bacteria of claim 4 in the synthesis of selenoproteins.

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