High-vitality glutathione peroxidase GPX3 (glutathione peroxidase) mutant and preparation method thereof

Abstract

The invention discloses a high-vitality glutathione peroxidase GPX3 (glutathione peroxidase) mutant and a preparation method thereof, belonging to the field of biotechnologies. With a human GPX3 as a template, through computer simulation and fixed point mutation, a novel high-vitality glutathione peroxidase GPX3 mutant is obtained, wherein the high-vitality glutathione peroxidase GPX3 mutant comprises 207 amino acids; and compared with the human GPX3, the high-vitality glutathione peroxidase GPX3 mutant does not contain cysteine and is higher in GPX activity and better in stability; all cysteines in the human GPX3 are mutated into serines or alanines, but the 54th site is always selenocysteine (SeCys). According to the method, the high-enzyme activity GPX3 mutant protein is directly expressed in a mammal cell strain or auxotroph prokaryotic expression system and an SPP (trisodium phosphate) low-temperature expression system thereof by using a genetic engineering technology. According to the preparation method, a novel extremely high GPX activity artificial enzyme can be prepared without chemical modification. The preparation method disclosed by the invention is wide in application prospect on the aspect of bio-pharmaceuticals.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a high-activity glutathione peroxidase GPX3 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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