Method for preparing recombinant glutathione peroxidase by use of eukaryotic secretory expression system

Abstract

The invention provides a method for preparing recombinant glutathione peroxidase (GPX) by use of a eukaryotic secretory expression system and belongs to the field of the biotechnology. The method comprises the steps of assembling a target gene and a selenocystine (SeCys) insertion sequence on a secretory mammalian cell expression vector first, next, assembling the SeCys insertion sequence in combination with protein 2 on an endocellular mammalian cell expression vector, performing cotransfection of the same mammalian cell strain to the two vectors, and finally, synthesizing GPX by use of the mammalian cells in the presence of sodium selenite. The method is simple; the recombinant GPX is high in activity and yield and good in stability; the reduction of yield and inactivation caused by inclusion body renaturation are avoided, and therefore, the problems of limited source and unstable properties of the natural GPX are solved.

Description

[0001] The patent application for the present invention is a divisional application. The invention title of the original application is "Preparation of recombinant glutathione peroxidase by genetic engineering method", the application date is April 24, 2013, and the application number is 201310142866.1. technical field [0002] The invention relates to a method for preparing recombinant glutathione peroxidase by using a genetic engineering method, belongs to the field of biotechnology, and relates to gene recombination technology, gene mutation technology and recombinant protein expression technology. 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 reduc...

AI Technical Summary

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 that another preparation method of human single-chain selenium-containing abzyme is to introduce a catalytic group using a defective prokaryotic expression system (Escherichia coli) and gene mutation, but it is limited to human single-chain selenium-containing abzyme Preparation of abzymes, excluding recombinant glutathione peroxidase (GPX) and other GPX mimetic enzymes

[0007] 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 enzyme activity

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