Highly active glutathione peroxidase gpx2 mutant and preparation method thereof

Abstract

The invention provides a glutathione peroxidase GPX2 mutant with high activity and a preparation method thereof, and belongs to the field of biotechnology. According to the invention, the artificial GPX2 is used as a template to obtain a novel glutathione peroxidase GPX2 mutant with high activity through computer simulation and site-specific mutagenesis. The glutathione peroxidase GPX2 mutant with high activity comprises 203 amino acids; compared with artificial GPX2, the glutathione peroxidase GPX2 mutant with high activity does not contain cysteine and has higher GPX activity and better stability and enables, and all cysteines in the artificial GPX2 are enabled to be mutated into serine, but selenocysteine (SeCys) still is in the 40th site. According to the preparation method, based on a genetic engineering technology, the GPX2 mutant protein is directly expressed in mammal cell strains or an auxotroph prokaryotic expression system and an SPP low-temperature expression system; the novel artificial enzyme with high GPX2 activity can be prepared without chemical modification; the method provided by the invention has a wide application perspective in bio-pharmaceuticals.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a high-activity glutathione peroxidase GPX2 mutant and a preparation method thereof. Background technique [0002] 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 agent to decompose hydrogen peroxide and various hydroperoxides in the body, so it can remove reactive oxygen species (ROS) in the body and prevent lipid peroxidation. Treat various diseases caused by active oxygen, such as aging, ultraviolet radiation, cardiovascular and cerebrovascular diseases, cataracts, tumors, etc. Different from other antioxidant enzymes, GPX can not only scavenge ROS, but also degrade l...

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Problems solved by technology

[0004] Chinese patents 94102481.4, 96112628.0, 99104234.4, and 200810050556.6 disclose various selenium-containing abzymes. The preparation method is exactly to apply the chemical mutation (modification) method to introduce the catalytic group SeCys of GPX in the antibody template protein, which has the following disadvantages: (1) During the preparation process, only the step of chemical mutation (modification) will lose 20-40% of the enzyme protein, resulting in a significant drop 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

[0005] Chinese patent 200810050556.6 also discloses another preparation method of human single-chain selenium-containing abzyme, which uses an auxotrophic prokaryotic expression system (Escherichia coli) and gene mutation 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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