High-activity glutathion peroxidase GPX 1 mutant and its preparation method

Abstract

A high-activity glutathion peroxidase GPX 1 mutant and its preparation method belong to the field of biotechnology. A mutant gene is obtained by a gene mutation or synthesis method, and then a catalytic group SeCys of GPX is introduced into a substrate binding part of the mutant through an auxotroph prokaryotic expression system or an auxotroph and SPP low-temperature combined expression system so as to endow the mutant with high GPX activity; or a target gene, along with a SeCys insertion sequence, is firstly assembled onto a secreting type mammalia cellular expression vector, and then a binding protein 2 of the SeCys insertion sequence is assembled onto an intracellular mammalia cellular expression vector, contransfection of the same lactation cell strain is carried out by the two vectors, and GPX is synthesized by the lactation cell in the presence of sodium selenite. The method provided by the invention is simple. The mutant provided by the invention has advantages of high activity, high yield and good stability. Yield decreasing and inactivation caused by renaturation of an inclusion body are avoided. Thus, natural GPX source limitation and unstable properties are solved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a high-activity glutathione peroxidase GPX1 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 that another preparation method of human single-chain selenium-containing abzyme is to use auxotrophic prokaryotic expression system (E. Preparation of selenium 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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