Glutathione peroxidase GPX2 mutant containing serine and preparation method of mutant

A glutathione peroxide and mutant technology, which is applied in the biological field, can solve the problems that the catalytic group cannot achieve the specificity of the gene mutation method, is not suitable for the direct expression of selenoproteins, and the yield of simulated enzymes is decreased, etc. Achieving the effect of avoiding yield drop and inactivation, high yield and not easy inactivation

Inactive Publication Date: 2015-05-27
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Chinese patent 94102481.4, 96112628.0, 99104234.4, the preparation method of all kinds of selenium-containing abzymes disclosed in 200810050556.6 is exactly to apply the chemical mutation (modification) method to introduce the catalytic group SeCys of GPX in the antibody class template protein, has the following disadvantages: (1) in In the preparation process, only the step of chemical mutation (modification) will lose 20-40% of the enzyme protein, resulting in a significant decline in the yield of the simulated enzyme; (2) the cycle of preparing the simulated enzyme is long, the operation is cumbersome, and the time is long; (3) In the process of chemical mutation, phenylmethylsulfonyl fluoride, acetonitrile, etc. need to be used, these substances are toxic substances; (4) lack of targeting, for large protein molecules, a chemical reaction is often introduced at different sites Multiple non-specific catalytic groups, so the introduction of catalytic groups by chemical mutation cannot achieve the specificity of gene m

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  • Glutathione peroxidase GPX2 mutant containing serine and preparation method of mutant
  • Glutathione peroxidase GPX2 mutant containing serine and preparation method of mutant
  • Glutathione peroxidase GPX2 mutant containing serine and preparation method of mutant

Examples

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Embodiment 1

[0031] Example 1: Preparation of genetically engineered human GPX2 mutant protein using a synthetic gene combined with SPP and auxotrophic prokaryotic expression system

[0032] According to the amino acid sequence of the GPX2 mutant described in Sequence 1 (SEQ ID No: 1) of the present invention, artificially synthesized in a biological company with a DNA synthesizer, it can be expressed in auxotrophic strains described in Sequence 1 (SEQ ID No: 1). The gene of the GPX2 mutant protein ensures that the 5' end of the GPX2 mutant gene contains a start codon (ATG) and an Nde I restriction site, and the 3' end contains a stop codon and a Hind III restriction site, and the GPX2 mutation The coding sequence TGA of No. 40 SeCys of the body gene was replaced by the codon (TGC) of Cys, and the full length of the gene did not contain the ACA sequence; the specific target gene sequence was the No. , 68, 77 and 105 cysteine ​​codons are replaced by serine coding sequence (in sequence: AGC...

Embodiment 2

[0036] The preparation method is exactly the same as in Example 1, except that the 117th alanine coding sequence is replaced with a serine codon (TCC) when synthesizing the coding gene of the GPX2 mutant, and the 117th of the finally obtained GPX2 mutant protein is Serine, not alanine, and other amino acid sequences are completely identical to the first method, that is, the GPX2 mutant protein described in Sequence 2 (SEQ ID No: 2) of the present invention. The molecular weight of the mutant has only slight changes, and there is no difference in the results of SDS-PAGE and Western Blot, see figure 1 Lane 9 of . But its GPX activity is 2417U / μmol, slightly higher than that of Example 1, reaching the same order of magnitude as natural GPX.

Embodiment 3

[0038] Except that the expression vector pCold III (TAKARA, Cat. #3369) used in Example 1 was replaced with pCold I (TAKARA, Cat. #3367) with a histidine purification tag, the rest were exactly the same as in Example 1, that is, the final The GPX2 mutant protein described in Sequence 3 (SEQ ID No: 3) of the present invention was obtained. The mutant introduces 16 exogenous amino acids including 6 histidines on the carrier at the amino terminal, so the molecular weight has increased, which can be seen on the results of SDS-PAGE and Western Blot, see figure 1 The advantage of lane 11 is that the target protein can also be purified with a nickel-affinity layer system. Its GPX activity is 2336U / μmol, which is slightly lower than Example 2, which proves that the purification tag has no obvious effect on the activity of the protein, and the activity reaches the same order of magnitude as natural GPX.

[0039]

[0040]

[0041]

[0042]

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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

[0001] This patent application is a divisional application of the Chinese patent "Highly active glutathione peroxidase GPX2 mutant and its preparation method". The filing date of the original application is: 2014-02-18, and the application number of the original application is: 201410054696.6, the publication number of the original application is: CN103756984A, and the publication date is: 2014-04-30. technical field [0002] The invention belongs to the field of biotechnology, and in particular relates to a serine-containing glutathione peroxidase GPX2 mutant and a preparation method thereof. Background technique [0003] The substrate of selenium-containing glutathione peroxidase (GPX) is glutathione (GSH), and the catalytic group is selenocysteine ​​(SeCys). In organisms, GPX together with superoxide dismutase (SOD) and catalase (CAT) constitute the body's antioxidant defense system. GPX plays an important role in this system. It uses the substrate GSH as a reducing agen...

Claims

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Application Information

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IPC IPC(8): C12N9/08
CPCC12N9/0065C12Y111/01009
Inventor 宋健魏景艳郭笑
Owner JILIN UNIV
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