CN104651329A - Glutathione peroxidase GPX2 mutant containing serine and preparation method of mutant - Google Patents

Abstract

The invention discloses a glutathione peroxidase GPX2 mutant containing serine and a preparation method of the mutant, and belongs to the field of biotechnology. The novel high-activity glutathione peroxidase GPX2 mutant is obtained by taking human GPX2 as a template through computer simulation and site-specific mutagenesis, and the mutant consists of 203 amino acids; compared with the human GPX2, the mutant is free from cysteine, and is relatively high in GPX activity and better in stability; all cysteine in the human GPX2 is mutated into serine, but the 40th site is still selenocysteine (SeCys), alanine (Ala) on the 117th-site is mutated into serine (Ser), and a formed sequence is as shown in SEQ ID No:2. The method can achieve the direct expression of GPX2 mutant protein having high enzymatic activity in mammalian cell lines or auxotroph prokaryotic expression system and SPP low-temperature expression system thereof by virtue of genetic engineering technology. The method can prepare a novel artificial enzyme having quite high GPX activity without chemical modification. The method disclosed by the invention has a broad application prospect in the aspect of biological pharmacy.

Description

A kind of Selenoperoxidase GPX2 mutant containing Serine and preparation method thereof

Present patent application is the divisional application of Chinese patent " high vigor Selenoperoxidase GPX2 mutant and preparation method thereof ", the applying date of original application is: 2014-02-18, the application number of original application is: 201410054696.6, the publication No. of original application is: CN103756984A, and date of publication is: 2014-04-30.

Technical field

The invention belongs to biological technical field, be specifically related to a kind of Selenoperoxidase GPX2 mutant containing Serine and preparation method thereof.

Background technology

Substrate containing the Selenoperoxidase (GPX) of selenium is gsh (GSH), and catalytic group is seleno-cysteine (SeCys).In vivo, the same superoxide-dismutase of GPX (SOD), catalase (CAT) together form body anti-oxidative defense system.GPX plays an important role in this system, it with substrate GSH for reductive agent, hydrogen peroxide in decomposer and all kinds of hydroperoxide, thus can active oxygen (ROS) in purged body, prevent lipid peroxidation, treat the various diseases caused by active oxygen, as aging, ultraviolet radiation, cardiovascular and cerebrovascular diseases, cataract, tumour etc.Different from other antioxidase, GPX is except removing except ROS, and lipid peroxide of can also degrading, prevents cellular superoxideization from damaging, and the function of the Cell protection of this uniqueness makes it in antioxidase system, occupy the position of particularly important.But, because the source of natural GPX is quite limited, poor stability, cause its artifact and the research of stand-in thereof to receive much concern.

Small molecule mimetics mainly contains PZ51 (ebselen), AL3823A, BXT series product, and their weakness is that vigor is low, about being only the thousandth of natural GPX.The GPX analogue enztme that macromolecular stand-in mainly contain abzyme (Chinese patent 94102481.4 and 96112628.0) and prepare for protein template with glutathione S-transferase (GST), its vigor is significantly higher than small molecule mimetics.Obviously, be that the macromole GPX analogue enztme of Template preparation have received better effect to have the albumen of gsh (GSH) combining site, therefore the GPX analogue enztme technology of preparing of these high vigor of exploitation preparation is with regard to the study hotspot of Cheng Liao academia, has broad application prospects for fields such as molecular biology, physiotechnology and medical science.The method that success has been developed so far has: the catalytic group seleno-cysteine (SeCys) introducing GPX with chemical method (Chinese patent 94102481.4,96112628.0) or auxotroph prokaryotic expression system (Chinese patent 200810050556.6) in non-GPX class template albumen.

Chinese patent 94102481.4,96112628.0,99104234.4, the preparation method of all kinds of selenium-containing abzyme disclosed in 200810050556.6 is exactly the catalytic group SeCys that applied chemistry sudden change (modification) method introduces GPX in antibody class template protein, there is following shortcoming: (1) is in preparation process, only chemical mutation (modification) this step will lose the zymoprotein of 20-40%, causes analogue enztme productive rate significantly to decline; (2) cycle of preparing analogue enztme is long, complex operation, and the time is long; (3) need to use phenylmethylsulfonyl fluoride, acetonitrile etc. in chemical mutation process, these materials are virose material; (4) lack targeting, for large protein molecular, chemical reaction different loci of being everlasting introduces multiple non-specific catalytic group, and therefore chemical mutation is introduced catalytic group and cannot be reached the such specificity of transgenation method.

Chinese patent 200810050556.6 also discloses the another kind of preparation method of people source strand selenium-containing abzyme, introduce catalytic group by auxotroph prokaryotic expression system (intestinal bacteria) and transgenation method, but it is only limitted to the preparation of people source strand selenium-containing abzyme, do not comprise the preparation method of Selenoperoxidase (GPX) mutant and other GPX analogue enztme.Have the glutathione S-transferase (GST) of GPX activity preparation method (Yu, H.J., Liu, J.Q., a., Li, J., Luo, G.M., and Shen, J.C.J.Biol.Chem.2005,280,11930-11935) also appear in the newspapers, although this method also adopts auxotroph prokaryotic expression system and transgenation method to introduce catalytic group, it is only limitted to be that template protein prepares GPX analogue enztme with GST, does not comprise with natural GPX for Template preparation GPX mutant.In non-GPX class template albumen, introducing with auxotroph prokaryotic expression system catalytic group prepares analogue enztme method is catalytic group is incorporated into it that there is same substrate GSH combining site with GPX to plant in albumen and make it produce GPX vigor.

But do not possess the catalytic group of natural GPX self due to these template protein, be therefore difficult to find ideal and the position of catalytic group accurately; Simultaneously owing to not resembling catalytic triads desirable natural GPX in these template protein, therefore the catalytic efficiency of this kind of analogue enztme is not high yet.If with natural GPX for template gene engineering method prepares GPX mutant, these shortcomings can be overcome.Because the codon UGA of the catalytic group SeCys of the GPX that encodes is terminator codon, in common prokaryotic expression system, need in the open reading frame of GPX gene, next-door neighbour SeCys codon UGA downstream introduce neck ring structure UGA could be translated into SeCys instead of terminator codon, and the introducing of neck ring will inevitably cause the change of GPX space conformation in open reading frame, and then affect enzymic activity.Therefore common prokaryotic expression system is unsuitable for directly expressing the Selenonic protein with GPX activity.

Summary of the invention

The object of the present invention is to provide a kind of Selenoperoxidase GPX2 mutant containing Serine and preparation method thereof.

Using gene engineering technique, cell culture technology, auxotroph prokaryotic expression technology and SPP (Single protein production, single protein production) system hypothermia expression technology prepares the GPX2 mutant with high GPX vigor, is in mammalian cell strain or auxotroph prokaryotic expression system and SPP low temperature expression system thereof, directly express the method with the GPX2 mutant protein of enzymatic activity high with genetic engineering technique.Present method does not need chemically modified to prepare to have the novel artificial enzyme of high GPX vigor.The inventive method has broad application prospects in bio-pharmaceuticals.

The present invention with people GPX2 (see NCBI, NM_002083.3) be template, rite-directed mutagenesis is fitted by computer mould, obtain a kind of novel high vigor Selenoperoxidase GPX2 mutant, it is made up of 203 amino acid, compared with people GPX2, it is not containing halfcystine, there is higher GPX vigor and better stability, it is that cysteine mutation all in people GPX2 are become Serine, but the 40th remains seleno-cysteine (SeCys, one-letter abbreviations is U, is write as Xaa in sequence table).The aminoacid sequence (SEQ ID No:1) of described GPX2 mutant is as follows:

MAFIAKSFYDLSAISLDGEKVDFNTFRGRAVLIENVASLUGTTTRDFTQLNELQSRFPRRLVVLGFPSNQFGHQENSQNEEILNSLKYVRPGGGYQPTFTLVQKSEVNGQNEHPVFAYLKDKLPYPYDDPFSLMTDPKLIIWSPVRRSDVAWNFEKFLIGPEGEPFRRYSRTFPTINIEPDIKRLLKVAI

Further, the concrete aminoacid sequence of described GPX2 mutant also comprises, and any one or more L-Ala (Ala) in above-mentioned aminoacid sequence is sported any one novel GPX2 mutant that Serine (Ser) is formed.Such as 117 L-Ala (Ala) are sported Serine (Ser), the sequence formed is SEQ ID No:2 (embodiment 2).

Further, the concrete aminoacid sequence of described GPX2 mutant also comprises, and introduces any one novel GPX2 mutant that Histidine purification tag on this secretor type protokaryon or carrier for expression of eukaryon and other amino acid etc. formed owing to employing the secretor type protokaryons such as the pColdI (TAKARA company) of being convenient to target protein purifying or carrier for expression of eukaryon at the aminoterminal of above-mentioned aminoacid sequence or carboxyl terminal.The sequence SEQ ID No:3 (embodiment 3) such as formed after the aminoterminal of sequence SEQ ID No:2 introduces the Histidine purification tag of pColdI (TAKARA company) prokaryotic expression carrier and the amino acid of factor Xa cleavage site.

Preparation of the present invention contains the method for the Selenoperoxidase GPX2 mutant of Serine; it is the gene first can expressing GPX2 mutant protein of the present invention at biotech firm's DNA synthesizer synthetic; guarantee in gene not containing ACA sequence; combine preparation GPX2 mutant protein with single protein production systems and auxotroph prokaryotic expression system again, concrete steps are as follows:

1), the structure of expression vector: according to the aminoacid sequence of GPX2 mutant of the present invention, the gene of GPX2 mutant protein can be expressed in auxotrophic strain at biotech firm's DNA synthesizer synthetic, guarantee that 5 ' end of target gene is containing initiator codon (ATG), 3 ' end is containing terminator codon, and specific restriction enzyme site is all contained at two ends, guarantee that the encoding sequence of 40 seleno-cysteines (SeCys) of target gene replaces with the codon (can be TGC etc.) of halfcystine (Cys), and full length gene is not containing ACA sequence, concrete gene order can be (see NCBI by GPX2 gene, NM_002083.3) in, the encoding sequence of halfcystine of the 55th, 68,77 and 105 replaces with the codon of Serine, the codon of the 40th seleno-cysteine replaces with the codon of halfcystine, the encoding sequence of the 117th L-Ala replaces with the codon of Serine, and according to the degeneracy of codon, under the prerequisite not changing aminoacid sequence, ACA sequences all in gene is all replaced with the encoding gene that non-ACA sequence obtains, also can be that other any one can express GPX2 mutant protein in auxotrophic strain, and the encoding gene (due to the degeneracy of codon, with amino acid sequence can have multiple different encoding gene) of total length not containing ACA sequence, contain GPX2 mutant gene and the secretor type prokaryotic expression carrier (as pCold series etc.) of specific restriction enzyme site with identical restriction endonuclease cutting two ends, then by specific restriction enzyme site, GPX2 mutant gene is assembled on secretor type prokaryotic expression carrier with DNA ligase, described specific restriction enzyme site can be contain in the multiple clone site of expression vector and any one restriction enzyme site non-existent in GPX2 mutant gene, be the intrinsic DNA sequence dna by the based composition of restriction endonuclease identification on carrier,

2), the screening of positive transformant and the expression and purification of albumen:

By step 1) the middle competent cell containing expression vector conversion auxotrophic strain-BL21 (DE3) Cys of GPX2 mutant gene built, be coated with the nutrient agar plate containing Cys, screening positive strain; Again by pMazF (TAKARA, Cat#3367) the Plastid transformation positive strain containing express nucleic acid restriction endonuclease MazF, screen positive transformant with containing Double M9 solid medium; Positive transformant is spread cultivation after supporting, in the substratum containing SeCys, essential growth factor and nutrient substance, through IPTG low temperature 4-25 DEG C of abduction delivering, auxotrophic strain-BL21 (DE3) Cys can synthesize SeCys with the codon of Cys, directly give expression to the GPX2 mutant containing SeCys at the combining site of substrate GSH, zymoprotein is expressed with soluble form and is secreted in the pericentral siphon chamber of thalline, and MazF is by identifying and cutting off single stranded RNA particular sequence ACA, suppress the expression of host protein; First cultivation screening high expression level strain in a small amount, then enlarged culturing and abduction delivering; Under collection, washing, ice bath, ultrasonication thalline, release zymoprotein, centrifugal by liquid cryogen, removes bacterial sediment, obtains supernatant liquor; With gsh (GSH) affinitive layer purification GPX2 mutant protein, after dialysis freeze-drying, namely obtain GPX2 mutant protein sterling.Target protein is identified with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Westernblot).

The method introduces catalytic group by inducing auxotroph prokaryotic expression system under low temperature at the substrate binding site of GPX2 mutant, thus produces the GPX2 mutant of high vigor.

Described uses gsh (GSH) affinitive layer purification GPX2 mutant protein, is by pH7.5,50mmol/L Tris-Cl balance and wash-out foreign protein, with the buffer solution elution target protein containing 10mmol/L GSH.

Described is centrifugal by liquid cryogen, can at 4 DEG C, the centrifugal 15-30min of 8000-12000g.

Further, use and be convenient to the secretor type protokaryons such as the pColdI (TAKARA company) of target protein purifying or carrier for expression of eukaryon and introduce at the aminoterminal of target protein or carboxyl terminal any one novel GPX2 mutant that Histidine purification tag on this carrier and other amino acid etc. formed.The sequence SEQ ID No:3 such as formed after the aminoterminal of sequence SEQ ID No:2 introduces the Histidine purification tag of pColdI (TAKARA company) prokaryotic expression carrier and the amino acid of factor Xa cleavage site.

The present invention has following characteristics:

(1) the present invention uses the genetic engineering technique such as simple gene chemical synthesis or amplification, transgenation and expression, and preparation method is simple.

(2) the GPX2 mutant protein vigor prepared of the inventive method is high, and reached the same order of magnitude of natural GPX vigor, Cell Biology Experiment has confirmed there is stronger provide protection to the myocardial cell of Hydroperoxide injury.

(3) GPX2 mutant protein of the present invention has superpower stability, not easy in inactivation, this feature supplements defect of natural GPX.

(4) the secreted expression carrier of the present invention's use, GPX2 mutant protein can be expressed with soluble form and be secreted into the external of colibacillary pericentral siphon chamber or cells of mamma animals, the targeting signal peptide of pilot protein secreting, expressing is excised automatically by thalline or cell, expressed albumen is solubility, there is the space conformation of native protein and the activity of Geng Gao, do not need renaturation, the productive rate that renaturing inclusion bodies process can be avoided to cause declines and inactivation, thus with short production cycle.

(5) the SPP low temperature expression system that the present invention uses can utilize the identification of MazF protease specificity and the characteristic of the mRNA of cutting containing ACA sequence, make thalline can only without the protein of ACA sequence in composite coding gene, therefore the albumen mainly foreign protein of new synthesis, thus raising productive rate and Cys are to the transformation efficiency of SeCys, therefore have the double dominant that vigor is high, productive rate is high.

(6) the eukaryotic expression that the present invention is used directly uses the selenocysteine insertion sequence (SECIS) of GPX self, does not need to introduce SECIS in addition and enters expression vector, and thus simple to operate and available conventional carrier for expression of eukaryon is implemented.

These advantages are all conducive to scale operation, for practical application from now on lays a solid foundation, solve the problem that natural GPX source is not enough, character is unstable and vigor is low, have broad application prospects in bio-pharmaceuticals.

Accompanying drawing explanation

Fig. 1: the SDS-PAGE of the GPX2 mutant protein that purifying obtains (on) and Western blot (under) result: wherein M is molecular weight of albumen Marker, and 1,2,3 swimming lanes are target proteins prepared by embodiment 1,2,3.

Embodiment

Embodiment 1: prepare genetically engineered people GPX2 mutant protein with the gene association SPP synthesized and auxotroph prokaryotic expression system

The aminoacid sequence of the GPX2 mutant according to sequence 1 (SEQ ID No:1) of the present invention, can the gene of GPX2 mutant protein in auxotrophic strain described in expressed sequence 1 (SEQ ID No:1) with DNA synthesizer synthetic in biotech firm, guarantee that 5 ' end of GPX2 mutant gene is containing initiator codon (ATG) and Nde I restriction enzyme site, 3 ' end is containing terminator codon and Hind III digestion site, the encoding sequence TGA of No. 40 SeCys of GPX2 mutant gene replaces with the codon (TGC) of Cys, and full length gene is not containing ACA sequence, concrete target-gene sequence is (see NCBI by GPX2 gene, NM_002083.3) in the 55th, 68, the codon of the halfcystine of 77 and 105 replaces with the encoding sequence of Serine (successively: AGC, AGC, AGT, AGT), the codon of the 40th seleno-cysteine replaces with the encoding sequence (TGC) of halfcystine, again according to the degeneracy of codon, under the prerequisite not changing aminoacid sequence, ACA series jumps all in GPX2 gene are fallen, obtain the GPX2 mutant gene without ACA sequence, guarantee the GPX2 mutant protein described in this gene encodes sequence 1 (SEQ ID No:1) of the present invention, and 5 ' of target gene end is containing initiator codon (ATG) and Nde I restriction enzyme site, 3 ' end is containing terminator codon and Hind III digestion site, after restriction endonuclease Nde I and Hind III double digestion, be connected on pCold III (TAKARA, Cat.#3369) carrier that same enzyme cuts.

Transform auxotrophic E. coli BL21 (DE3) Cys with the carrier (pCold III-GPX2 dashes forward) that GPX2 mutant gene is housed, be coated with the nutrient agar plate containing Cys, screening positive strain.Again the plasmid pMazF (TAKARA, Cat.#3369) of express nucleic acid restriction endonuclease MazF is transformed in this positive strain.Bacterium liquid is uniformly coated in the M9-CAA solid medium containing 100 μ g/mL Amp, 25 μ g/mLKana and 25 μ g/mL and screens positive transformant.Positive transformant is inoculated in 1.2L M9 defect express in substratum (adding 100 μ g/ml penbritins, 50 μ g/ml kantlex, 50 μ g/ml Cys and each 100 μ g/ml of 19 seed amino acids except Cys in M9 substratum) to OD600 be 0.5.Bacterium liquid is placed in-20 DEG C of refrigerator 5min, bacterium liquid is cooled rapidly, afterwards bacterium liquid is placed in 15 DEG C of relaying persistent oscillations and cultivates 45min.15 DEG C of centrifugal 5min of 5000rpm, abandon supernatant.With the resuspended bacterial sediment of 0.9%NaCl, 15 DEG C of centrifugal 5min of 5000rpm, abandon supernatant, repeat this step.Then productive culture base (adding the 19 seed amino acid each 50-400 μ g/ml except Cys in M9 substratum) resuspended thalline is used, to add final concentration be 1mmol/L IPTG and final concentration is 200 μ g/mL SeCys, 15 DEG C of shaking culture 16-72h, GPX2 mutant protein is expressed in the pericentral siphon chamber of thalline with soluble form, and MazF is by identifying and cutting off single stranded RNA particular sequence ACA, suppress the expression of host protein.Collect thalline (6000rpm, 10min) and wash twice with isopyknic bufferT (50mM Tris buffer, pH7.5, containing 1mM EDTA).With the resuspended bacterial sediment of BufferT, add the PMSF (phenylmethylsulfonyl fluoride) of final concentration 1mM, ultrasonication thalline, 4 DEG C, the centrifugal 30min of 12000rpm, collect supernatant.By specification process GSH affinity post, application damping fluid (50mmol/L Tris, pH7.5) balance, adds on GSH affinity post, with level pad wash-out foreign protein, with 10mmol/L GSH wash-out target protein by supernatant liquor.By elutriant dialysis also freeze-drying, obtain people GPX2 mutant protein.Target protein is identified with denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Western blot), its molecular weight is 20.9kDa, and be combined with anti-GPX2 antibodies specific, prove to have successfully been obtained target protein, see the 1st swimming lane of Fig. 1.Its GPX vigor is 2636U/ μm of ol, reaches the same order of magnitude of natural GPX.

The SPP low temperature expression system that the present embodiment uses can utilize the identification of MazF protease specificity and the characteristic of the mRNA of cutting containing ACA sequence, make thalline can only without the protein of ACA sequence in composite coding gene, therefore the albumen mainly foreign recombinant proteins of new synthesis, thus raising productive rate and Cys are to the transformation efficiency of SeCys, therefore have high high yield double dominant of living.

Embodiment 2:

Preparation method is identical with embodiment 1, when being the encoding gene of synthesis GPX2 mutant, the encoding sequence of the 117th L-Ala is replaced with the codon (TCC) of Serine, the 117th of the GPX2 mutant protein finally obtained is Serine, instead of L-Ala, other aminoacid sequence is identical with first method, the GPX2 mutant protein namely described in sequence 2 (SEQ ID No:2) of the present invention.This mutant molecule amount only has small change, as broad as long in SDS-PAGE and Western Blot result, sees the 9th swimming lane of Fig. 1.But its GPX vigor is 2417U/ μm of ol, a little more than embodiment 1, reach the same order of magnitude of natural GPX.

Embodiment 3:

Remove expression vector pCold III (TAKARA used for embodiment 1, Cat.#3369) pColdI (TAKARA of band Histidine purification tag is changed into, Cat.#3367), all the other are identical with embodiment 1, namely obtain the GPX2 mutant protein described in sequence 3 (SEQ ID No:3) of the present invention the most at last.This mutant introduces 16 exogenous amino acids carrier comprising 6 Histidines at aminoterminal, therefore molecular weight increases to some extent, SDS-PAGE and Western Blot result can be seen, see the 11st swimming lane of Fig. 1, its advantage also can use the affine series of strata target protein of nickel.Its GPX vigor is 2336U/ μm of ol, and a little less than embodiment 2, prove that the vigor of purification tag to albumen does not have a significant effect, vigor reaches the same order of magnitude of natural GPX.

Claims (4)

1. the Selenoperoxidase GPX2 mutant containing Serine, is characterized in that: its aminoacid sequence is as shown in SEQID No:2.

2. a kind of Selenoperoxidase GPX2 mutant containing Serine as claimed in claim 1, is characterized in that: its aminoacid sequence is as shown in SEQ ID No:3.

3. the preparation method of a kind of Selenoperoxidase GPX2 mutant containing Serine according to claim 1, its step is as follows:

1), the structure of expression vector: according to the aminoacid sequence of GPX2 mutant of the present invention, the gene of GPX2 mutant protein can be expressed in auxotrophic strain at biotech firm's DNA synthesizer synthetic, guarantee that 5 ' end of target gene is containing initiator codon ATG, 3 ' end is containing terminator codon, and specific restriction enzyme site is all contained at two ends, guarantee that the encoding sequence of 40 seleno-cysteine SeCys of target gene to replace with in the codon of halfcystine Cys the 55th, 68, the encoding sequence of the halfcystine of 77 and 105 replaces with the codon of Serine, the codon of the 40th seleno-cysteine replaces with the codon of halfcystine, the encoding sequence of the 117th L-Ala replaces with the codon of Serine, and according to the degeneracy of codon, under the prerequisite not changing aminoacid sequence, ACA sequences all in gene is all replaced with the encoding gene that non-ACA sequence obtains, or other any one can express GPX2 mutant protein in auxotrophic strain, and the encoding gene of total length not containing ACA sequence, contain GPX2 mutant gene and the secretor type prokaryotic expression carrier of specific restriction enzyme site with identical restriction endonuclease cutting two ends, then by specific restriction enzyme site, GPX2 mutant gene is assembled on secretor type prokaryotic expression carrier with DNA ligase, described specific restriction enzyme site be contain in the multiple clone site of expression vector and any one restriction enzyme site non-existent in GPX2 mutant gene, be the intrinsic DNA sequence dna by the based composition of restriction endonuclease identification on carrier,

2), the screening of positive transformant and the expression and purification of albumen:

By step 1) the middle competent cell containing expression vector conversion auxotrophic strain-BL21 (DE3) Cys of GPX2 mutant gene built, be coated with the nutrient agar plate containing Cys, screening positive strain; Again by the pMazF Plastid transformation positive strain containing express nucleic acid restriction endonuclease MazF, screen positive transformant with containing Double M9 solid medium; Positive transformant is spread cultivation after supporting, in the substratum containing SeCys, essential growth factor and nutrient substance, through IPTG low temperature 4-25 DEG C of abduction delivering, auxotrophic strain-BL21 (DE3) Cys can synthesize SeCys with the codon of Cys, directly give expression to the GPX2 mutant containing SeCys at the combining site of substrate GSH, zymoprotein is expressed with soluble form and is secreted in the pericentral siphon chamber of thalline, and MazF is by identifying and cutting off single stranded RNA particular sequence ACA, suppress the expression of host protein; First cultivation screening high expression level strain in a small amount, then enlarged culturing and abduction delivering; Under collection, washing, ice bath, ultrasonication thalline, release zymoprotein, centrifugal by liquid cryogen, removes bacterial sediment, obtains supernatant liquor; With gsh GSH affinitive layer purification GPX2 mutant protein, after dialysis freeze-drying, namely obtain GPX2 mutant protein sterling; Described uses gsh GSH affinitive layer purification GPX2 mutant protein, is by pH7.5,50mmol/LTris-Cl balance and wash-out foreign protein, with the buffer solution elution target protein containing 10mmol/L GSH; Described is centrifugal by liquid cryogen, is at 4 DEG C, the centrifugal 15-30min of 8000-12000g.

4. the preparation method of a kind of Selenoperoxidase GPX2 mutant containing Serine according to claim 2, is characterized in that: be formation sequence SEQ ID No:3 after the aminoterminal of sequence SEQ ID No:2 introduces the Histidine purification tag of pColdI prokaryotic expression carrier and the amino acid of factor Xa cleavage site.