Seryl tRNA synthetase mutant and application thereof

Abstract

The invention discloses a seryl tRNA synthetase mutant and application thereof, and belongs to the field of zymoprotein.The seryl tRNA synthetase mutant is characterized in that an interaction interface of SerRS and tRNASec is predicted through machine learning technologies such as AlphaFld3, so that key amino acid residues influencing tRNASec recognition in SerRS are accurately positioned, a semi-rational mutation strategy is adopted, the key sites of SerRS are directionally modified, and the seryl tRNA synthetase mutant is obtained. According to the present invention, the serRS variant with high tRNASec aminoacylation activity is constructed by using the serRS as the template, such that the Sec supply level is improved, the high-efficiency and high-fidelity expression of the selenoprotein is achieved, the false doping of the Ser is completely eradicated, and the new technical means is provided for the biosynthesis of the selenoprotein.

Description

Technical Field

[0001] The invention belongs to the field of enzyme proteins, and specifically relates to a seryl tRNA synthetase mutant and an application thereof. Background Art

[0002] Selenium is an essential trace element for the human body. It mainly exists in the form of selenocysteine ​​(Sec) and participates in a variety of physiological processes, including anti-oxidation, signal transduction and immune regulation. The active center of selenoproteins is usually composed of Sec. Compared with cysteine ​​(Cys), Sec has a stronger ability to resist oxidative inactivation. Therefore, selenoproteins play an important role in preventing major diseases such as cardiovascular disease, diabetes and cancer. However, common inorganic selenium products are difficult to be absorbed and utilized by the human body. There are almost no commercially available selenoprotein products on the market. The use of microbial cell factories to heterologously express proteins is a common protein production method. Some bacteria naturally have a synthesis and incorporation system for Sec. For example, Escherichia coli, which is commonly used in amino acid fermentation and protein expression, has been used to express bacterial and human selenoproteins. In these bacteria, the synthesis of Sec uses serine (Ser) as a precursor. However, unlike other amino acids, the synthesis reaction of Sec must be carried out in an aminoacylated form. The first step is to load Ser onto Sec's tRNA (tRNA Sec ). The reaction is catalyzed by seryl synthetase (SerRS), which can simultaneously recognize the tRNA (tRNA Ser ) and tRNA Sec Ser is loaded onto two tRNAs to generate their own aminoacyl tRNAs. The former is responsible for introducing Ser into the peptide chain during translation, while the latter converts the seryl hydroxyl group on it into a selenol group under the action of the enzyme to generate the aminoacylated Sec-tRNA. Sec , and then used for Sec incorporation. Due to the difference in tRNA structure, SerRS responsible for aminoacylation is more likely to recognize Ser tRNA, while Sec The aminoacylation efficiency of tRNA Ser 1%. Despite overexpression of tRNA Sec This bottleneck can be alleviated, but it can only increase the final Sec synthesis level by about 10%, mainly due to the interaction between SerRS and tRNA Sec In contrast, the catalytic enzymes in the subsequent pathways have a low affinity for tRNA Sec The match is extremely high and has the ability to convert Ser-tRNA above physiological concentration levels Sec Therefore, tRNA Sec The aminoacylation of tRNA has become the biggest rate-limiting link in the synthesis of Sec and selenoproteins. SecThe affinity of selenoprotein can significantly improve the synthesis efficiency of selenoprotein, thus providing a more effective strategy for solving the problem of selenium deficiency.

[0003] MUKAI T, SEVOSTYANOVAA, SUZUKI T, et al. A facile method for producing selenocysteine-containing proteins [J]. Angew Chem Int Ed Engl, 2018, 57(24): 7215-9. doi: 10.1002 / anie.201713215 discloses that tRNA Sec , tRNA Ser , or a type of allo-tRNA with a long acceptor arm and a short T arm structure as a blueprint, by replacing and fine-tuning the domains in the above tRNAs that bind to the elongation factor, artificial tRNAs such as tRNASecUX that can be recognized by the universal elongation factor EF-Tu were obtained, so that the artificial tRNA that can be loaded with Sec can autonomously complete decoding like other types of tRNA, and realize the incorporation of Sec at any site. However, the EF-Tu type elongation factor in this document will cause a large amount of Ser to be misincorporated, and the incorporation rate is low, resulting in low purity of the final synthesized selenoprotein. At the same time, this type of artificial tRNA still cannot efficiently bind to SerRS, and the expression level of selenoproteins is always affected by aminoacyl tRNA. Sec Limited by insufficient 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 disclosed that under the guidance of structural modeling and chloramphenicol acetyltransferase activity screening, three Ser-tRNA synthetases were evolved to improve serylation of selenocysteine ​​tRNAs. Sec The synthetic SerRS variants are:

[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] Among them, for example, K86C means that the lysine at position 86 of the amino acid sequence of the SerRS enzyme protein is mutated to cysteine. Compared with the wild-type SerRS, when using artificially modified tRNA as a substrate, these three SerRS mutants showed 10-fold, 8-fold and 4-fold increased k cat / K M However, this document is aimed at the modification of engineered tRNA unfoldase, and does not solve the problem of Ser misincorporation caused by the use of artificial tRNA. It only proves that the aminoacylation catalytic constant value of SerRS variant for artificial tRNA is improved, but does not actually prove that the expression of selenoprotein is improved. Sec synthesis and selenoprotein expression are still 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 amount, enzyme activity and purity of selenoprotein.

[0010] The technical solution of the present invention is: a seryl tRNA synthetase mutant, which is obtained by mutating any one of a to d or any combination of d and a, b or c on the basis of the seryl tRNA synthetase shown in SEQ ID NO.1:

[0011] a. The 229th amino acid mutated from threonine to arginine;

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

[0013] c. The amino acid at position 363 mutated from tryptophan to phenylalanine;

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

[0015] A gene encoding the above-mentioned seryl-tRNA synthetase mutant.

[0016] An expression vector containing the gene described above.

[0017] An engineered bacterium containing the above-mentioned expression vector.

[0018] The use of the mutants or genes or expression vectors or engineered bacteria described above in synthesizing selenoproteins.

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

[0020] In order to improve the synthesis and incorporation efficiency of Sec and increase selenoprotein synthesis, the present invention provides a SerRS mutant enzyme with higher catalytic efficiency, which can effectively improve the expression, enzyme activity and purity of selenoproteins. The biosynthesis of selenoproteins depends on the effective incorporation of selenocysteine ​​(Sec), while the synthesis of Sec and tRNA loading are limited by the catalytic efficiency of SerRS. The aminoacylation reaction catalyzed by SerRS is the key rate-limiting step. Sec The aminoacylation efficiency of tRNA Ser 1% is due to tRNA Sec With tRNA Ser There are significant differences in secondary structure and sequence, resulting in SerRS to tRNA Sec The recognition and binding ability of SerRS is weak. The present invention uses machine learning technology such as AlphaFold3 to predict SerRS and tRNA Sec The interaction interface of SerRS can precisely locate the tRNA Sec The key amino acid residues identified were targeted by a semi-rational mutation strategy to modify the key sites of SerRS and enhance its ability to regulate tRNA Sec The binding affinity and catalytic efficiency of tRNA Sec SerRS variants with aminoacylation activity can improve the supply level of Sec, achieve efficient and high-fidelity expression of selenoproteins, eliminate the misincorporation of Ser, and provide a new technical means for the biosynthesis of selenoproteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Alphafold 3 simulation prediction, where (a) Alphafold 3 predicted SerRS-tRNA Ser Complex structure; (b) Alphafold 3 predicted SerRS-tRNA Sec Composite structure.

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

[0023] Figure 3Single-bacteria screening of the SerRS_T382 mutant library shows the growth of some numbered bacteria under screening pressure; the mutation library containing different mutation directions of the T382 site of SerRS was screened. Under the same screening conditions, the growth rate of the strains expressing most of the T382 variants was significantly accelerated compared with the strains expressing the wild-type SerRS, indicating that the catalytic efficiency of the T382 site variants of SerRS is significantly improved.

[0024] Figure 4 Single-bacteria screening of the SerRS_W363 mutant library shows the growth of some numbered bacteria under screening pressure; the mutant library containing different mutation directions of the W363 site of SerRS was screened. Under the same screening conditions, the growth rate of the strain expressing most of the W363 variants was significantly accelerated compared with the strain expressing the wild-type SerRS, indicating that the catalytic efficiency of the W363 site variant of SerRS is significantly improved.

[0025] Figure 5 Growth of SerRS variant T382A and SerRS wild-type overexpressing strain under screening pressure; under the same screening conditions, the growth rate of the strain expressing SerRS variant T382A was significantly accelerated compared with the strain expressing wild-type SerRS, indicating that the catalytic efficiency of SerRS variant T382A was significantly improved.

[0026] Figure 6 Growth of SerRS mutant T229R, SerRS wild-type overexpressing strain and blank control strain under screening pressure; under the same screening conditions, the growth rate of the strain expressing SerRS variant T229R was significantly accelerated compared with the strain expressing wild-type SerRS, indicating that the catalytic efficiency of SerRS variant T229R was significantly improved.

[0027] Figure 7 Growth of SerRS mutant D300V, SerRS wild-type overexpressing strain and blank control strain under screening pressure; under the same screening conditions, the growth rate of the strain expressing SerRS variant D300V was significantly accelerated compared with the strain expressing wild-type SerRS, indicating that the catalytic efficiency of SerRS variant D300V was significantly improved.

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

[0029] Fig. 9 Growth of SerRS variant W363F:T382V and SerRS wild-type overexpressing strain under screening pressure; under the same screening conditions, compared with the strain expressing wild-type SerRS, the growth rate of the strain expressing SerRS variant W363F:T382V was significantly accelerated, indicating that the catalytic efficiency of SerRS variant W363F:T382V was significantly improved.

[0030] Fig.10 Growth of SerRS variant T229R:T382S and SerRS wild-type overexpressing strain under different screening pressures; different Carb and Serine concentrations are set in this figure. Under the conditions of adding 45 mg / L Carb, 1 g / L Ser, the growth rate of the strain expressing SerRS variant T229R:T382S is significantly accelerated compared with the strain expressing wild-type SerRS, indicating that the catalytic efficiency of SerRS variant T229R:T382S is significantly improved.

[0031] Fig.11 Using the SerRS mutant T382A of the present invention to express the mouse selenoprotein-glutathione peroxidase can significantly increase the expression amount of total protein and full-length selenoprotein compared with overexpression of only wild-type SerRS.

[0032] Fig.12 The mass spectrometry identification results of the mouse selenoprotein-glutathione peroxidase expressed using the SerRS mutant T382A of the present invention.

[0033] Fig.13 The use of the SerRS mutant T382A of the present invention can significantly increase the expression level of selenoformate dehydrogenase FdhH. DETAILED DESCRIPTION

[0034] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0035] Example 1 Experimental method

[0036] The biosynthesis of selenoproteins depends on the efficient incorporation of selenocysteine ​​(Sec), while the synthesis of Sec and tRNA loading are limited by the catalytic efficiency of SerRS. The aminoacylation reaction catalyzed by SerRS is the key rate-limiting step. Sec The aminoacylation efficiency of tRNASer is only 1% of that of tRNASer. Sec With tRNA Ser There are significant differences in secondary structure and sequence, resulting in SerRS to tRNA Sec The recognition and binding ability of SerRS is weak. The present invention uses machine learning technology such as AlphaFold3 to predict SerRS and tRNA Sec The interaction interface of SerRS can precisely locate the tRNA Sec The key amino acid residues identified were targeted by a semi-rational mutation strategy to modify the key sites of SerRS and enhance its ability to regulate tRNA Sec The binding affinity and catalytic efficiency of tRNA Sec The SerRS variant with aminoacylation activity is described in detail as follows:

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

[0038] This scheme establishes a rational design mutation library through two prediction methods, one is the classical molecular docking prediction method, and the other is the Alphafold 3 prediction method. The modeling method establishes a three-dimensional interaction model based on the amino acid sequence of the SerRS protein and the base sequence of the tRNA to analyze the interaction site between SerRS and tRNA. The SerRS used in the present invention is the background serS gene expression of the Escherichia coli MG1655.K12 strain, and the Gene ID in Genbank is 945506.

[0039] Classical molecular docking simulation prediction: based on tRNA of Escherichia coli MG1655 Sec Base sequence and 2D structure (RNAcentral ID:URS00002F9CDD_511145), tRNA prediction using simRNA2.0 tool Sec The three-dimensional structure of (https: / / genesilico.pl / SimRNAweb) was constructed using the default parameters of the website. SecAfter the three-dimensional structure, the three-dimensional structure of SerRS of Escherichia coli (PDB ID: 6R1M) was downloaded, and the water molecules and small molecule ligands in the SerRS molecular structure were deleted using Discovery studio 4.5. The docking of the two molecules was predicted using Autodock Vina. The docking parameters were 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, 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 tRNA of Escherichia coli MG1655 Sec The base sequence (RNAcentral ID:URS00002F9CDD_511145) and the protein sequence of Escherichia coli SerRS (PDB ID:6R1M) were used to predict two tRNAs using Alphafold3 Sec The complex structure with the SerRS protein dimer (https: / / golgi.sandbox.google.com / ). A total of three molecules were added for docking, including two RNA molecules, both of which were tRNA Sec Base sequence, and a SerRS protein dimer, two SerRS protein sequences were input as dimer protein sequences in turn, and the prediction parameters were all default parameters of the website. Finally, further analysis was performed based on the prediction model with the highest score (default is model_0).

[0041] (2) Establishment of mutation library:

[0042] Analysis of interaction regions and establishment of rational mutation library: for the obtained tRNA Sec The three-dimensional structure of the complex with SerRS was screened by Pymol 3.0 for the interaction between SerRS and tRNA Sec The amino acid residue sites with intermolecular forces were simultaneously screened for the SerRS molecules that interacted with tRNA Sec For amino acid residue sites with an intermolecular distance of less than 4 angstroms, a variety of mutation libraries were established for these sites that have the potential to enhance the catalytic ability of SerRS in theoretical analysis.

[0043] Semi-rational mutation library: The amino acid residue sites obtained by the two screening methods are taken as the union, and the single-point saturation mutation library is constructed respectively (the mutation direction of the original amino acid site is 20 kinds of classical amino acids). At the same time, for the screening sites with a distance of less than 150bp on the gene sequence, a multi-point combination saturation mutation library (multiple sites simultaneously contain the mutation directions of 20 kinds of classical amino acids) and a multi-point combination random primer mutation library are constructed respectively (the original base ratio of the amino acid site is 85%, and the remaining three bases are 5% each, to control the mutation rate and protect the normal function of the protein).

[0044] Establishment of 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‰ bases in the entire SerRS gene undergo non-directional mutations, and the locations of these mutations are completely random).

[0045] (3) Construction of screening system

[0046] This protocol uses a screening method based on carbenzyl antibiotics, and the growth of strains in an environment with carbenzyl antibiotics added indicates changes in the catalytic effect of SerRS. β-lactamase can cleave carbenzyl antibiotics. Strains that correctly express the enzyme can grow in an environment containing carbenzyl antibiotics, and vice versa. Some β-lactamases contain Cys residues that can form disulfide bonds, which are essential for maintaining enzyme activity. Such Cys residues can theoretically be replaced by Sec with similar properties to form selenosulfur bonds or diselenide bonds. When mutated to other amino acid residues, the enzyme structure will be destroyed and the strain will not be able to be resistant to carbenzyl antibiotics ( Figure 2 In other words, the gene can specifically distinguish Sec from other amino acids and thus 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 replaced with UAG in sequence to construct an expression plasmid. Sec CUA The expression plasmids of the two genes were co-transfected into the C321.ΔAΔselACDΔfdhFΔbla strain, and the strain overexpressing wild-type β-lactamase was used as a control. The growth level of the strain was detected in LB medium containing an appropriate concentration of carbenicillin to determine the codon substitution frequency that can lead to a significant decrease in bacterial concentration. On this basis, 0.5, 1.0 and 2.0 g / L of L-serine were added to the culture medium to create Ser-tRNA Sec CUAThe concentration gradient of L-serine was used to detect the growth level of bacteria. The increase in bacterial concentration with the addition of L-serine can be used to indicate tRNA Sec Increased aminoacylation level ( Figure 2 (b)

[0048] (4) Iterative screening:

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

[0050] First, perform a preliminary screening under a relatively low concentration of carbenzyl antibiotic screening pressure to screen for effective SerRS mutants. For the SerRS mutant library bacterial solution that can grow normally under the initial screening conditions, dilute and spread on the plate to obtain single colonies (the bacterial solution may contain a variety of SerRS mutants, and each single colony contains only one mutant), and each single colony is inoculated into a higher concentration of carbenzyl antibiotic environment, and rescreened under more stringent screening conditions to screen for SerRS mutants with obvious advantages.

[0051] After the screening, the SerRS mutants with obvious advantages in the rescreening were verified, and SerRS variants with the same mutation site were artificially constructed and overexpressed in the strain. If the strain overexpressing the SerRS variant grows faster than the strain overexpressing the wild-type SerRS under the screening conditions, it means that the mutation site of the dominant variant can affect the interaction between SerRS and tRNA. Sec affinity between tRNA Sec The rate of aminoacylation is increased.

[0052] (5) Synthesis of selenoproteins:

[0053] Formate dehydrogenase FdhH and glutathione peroxidase GPx are important selenoproteins, and the catalytic active center of both is Sec. FdhH or GPx was heterologously expressed in a strain overexpressing a SerRS variant, the protein was extracted and purified, the protein expression level was tested by SDS-PAGE, and protein spectrum was used to analyze whether Sec was correctly incorporated to verify the purity of the selenoprotein synthesized by this scheme. At the same time, enzyme activity was tested to verify the activity of the expressed protein.

[0054] Through the above screening, the SerRS mutants T229R; D300V; W363F; T382A; T382V; T382S were obtained.

[0055] Example 2 Verification of the advantages of the SerRS variant of the present invention over wild-type SerRS

[0056] The validation experiment usually sets up 2 or 3 groups, one of which expresses wild type (WT) SerRS and the other expresses SerRS variants. For example, SerRS:T229R indicates that the mutation site of the variant is threonine (T) at position 229, and the mutation direction is to arginine (R). sel-control indicates a blank control group that does not express SerRS. The screening pressure of the validation experiment is mainly adjusted by the concentration of the antibiotic carbenicillin sodium (Carb) and the replenishment concentration of serine (Serine, Ser). The higher the catalytic efficiency of the SerRS variant, the higher the efficiency of Sec synthesis and incorporation, the more selenoprotein β-lactamase synthesized by the bacteria, and the selenoprotein can cleave carbenicillin, allowing the strain to survive under the screening pressure of carbenicillin.

[0057] The Carb concentration is usually set at 30mg / L-60mg / L. Within this range, the higher the Carb concentration, the greater the screening pressure on the SerRS catalytic efficiency. The Ser concentration is usually set at 0.1g / L-4g / L. Within this range, Ser is one of the precursor reactants of the SerRS catalytic reaction. The higher its replenishment concentration, the more it can release the SerRS function and help the SerRS catalytic reaction proceed. 600 It refers to the optical density value measured when the wavelength of the spectrophotometer is set to 600nm. It is one of the commonly used methods for measuring bacterial quantity and can indirectly reflect the bacterial concentration in the liquid culture medium. 600 The higher the concentration, the more cells there are in the bacterial solution, and vice versa. Under certain concentrations of Carb and Ser, the growth of E. coli carrying the SerRS expression plasmid indicates the catalytic efficiency of SerRS. At the same time, the faster the bacterial growth or the greater the bacterial density, the higher the catalytic efficiency of SerRS expressed by the bacteria. According to the SerRS-tRNA simulation structure, amino acid sites that may interact are selected, and saturation mutations are performed on these sites to establish a single-point saturation mutation library containing 20 different amino acid mutation directions at the site. The mutant library is expressed in E. coli and initially screened in a culture medium containing 30 mg / L Carb. Compared with strains expressing only wild-type SerRS, strains expressing SerRS mutant libraries such as T382 grow faster, indicating that T382 ( Figure 3 ), W363( Figure 4 ), T229( Figure 6 ) and D300( Figure 7 ) site can improve the efficiency of Sec synthesis and incorporation.

[0058] SerRS variants that improved Sec synthesis and incorporation were screened in E. coli and their effects were verified in a medium containing 60 mg / LCarb. Compared with 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 Verification of the effect of SerRS combined mutation

[0060] The combined mutants of SerRS were expressed in Escherichia coli and their effects were verified in a medium containing 60 mg / L Carb. Compared with the strain expressing wild-type SerRS, the strains expressing the combined variants of SerRS 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 , Fig. 9 , Fig.10 ).

[0061] Example 4SerRS variants can increase selenoprotein expression

[0062] The SerRS mutant T382A obtained by the present invention was used in combination with other elements necessary for Sec incorporation to express and purify the mouse selenoprotein, glutathione peroxidase, in Escherichia coli MG1655. SDS-PAGE identification showed that compared with overexpression of wild-type SerRS, the SerRS mutant of the present invention can significantly increase the expression of selenoprotein ( Fig.11 , Fig.12 ).

[0063] Example 5SerRS variants can improve the enzyme activity and purity of selenoproteins

[0064] Using the SerRS mutant T382A obtained by the present invention, in combination with other elements necessary for Sec incorporation, the purified mouse selenoprotein-glutathione peroxidase was expressed in Escherichia coli MG1655, which can significantly increase the enzyme activity of selenoprotein. Using the DTNB method, compared with overexpression of wild-type SerRS, the present invention can increase the protein yield and enzyme activity of selenoprotein (Table 1).

[0065] Table 1 The use of the SerRS mutants of the present invention can improve the enzyme activity and purity of selenoproteins

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

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

[0068] Using the SerRS mutant T382A obtained by the present invention, in combination with other elements necessary for Sec incorporation, the selenoformate dehydrogenase FdhH was expressed and purified in Escherichia coli MG1655, which can significantly increase the protein expression level ( Fig.13 ).

Claims

1. A seryl-tRNA synthetase mutant, characterized in that: It is obtained by mutating any one of a to d or any combination of d and a, b or c based on the seryl tRNA synthetase shown in SEQ ID NO.1: a. The 229th amino acid mutated from threonine to arginine; b. The 300th amino acid mutated from aspartic acid to valine; c. The amino acid at position 363 mutated from tryptophan to phenylalanine; d. The amino acid at position 382 is mutated from threonine to alanine, valine or serine.

2. A gene encoding the seryl-tRNA synthetase mutant according to claim 1.

3. An expression vector containing the gene according to claim 2.

4. An engineered bacterium containing the expression vector according to claim 3.

5. Use of the mutant according to claim 1, the gene according to claim 2, the expression vector according to claim 3, or the engineered bacteria according to claim 4 in synthesizing selenoproteins.

Images