Glutathione peroxidase GPX2 mutant containing serine and preparation method of mutant

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...

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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 mutation method

[0006] Chinese patent 200810050556.6 also discloses another preparation method of human single-chain selenium-containing abzyme, which uses auxotrophic prokaryotic expression system (Escherichia coli) and gene mutation method to introduce catalytic groups, but it is limited to human single-chain Preparation of selenium-containing abzymes, excluding glutathione peroxidase (GPX) mutants and other GPX mimetic enzymes

Since the codon UGA of SeCys, the catalytic group of GPX, is a stop codon, in the common prokaryotic expression system, it is necessary to introduce a neck loop structure in the open reading frame of the GPX gene, immediately downstream of the codon UGA of SeCys, to translate UGA into SeCys instead of a stop codon, and the introduction of a neck loop in the open reading frame will inevitably cause a change in the spatial conformation of GPX, thereby affecting the enzymatic activity

Therefore, the common prokaryotic expression system is not suitable for direct expression of selenoproteins with GPX activity

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