Method of shearing fusion protein by escherichia coli intracellular protease

Abstract

The invention relates to a method of separating a target protein by shearing a fusion protein by escherichia coli intracellular protease. Used host bacteria is obtained by integrating a TEV (tobacco etch virus) protease gene driven by a strong promoter T7 to escherichia coli expression host bacteria BL21(DE3) by virtue of a recombinant engineering method. An expression vector is obtained by cloning maltose-binding protein and stuffer fragment to the expression vector pET30(+). A target gene not containing a terminator replaces the stuff segment to obtain a target gene fusion expression vector, and the tail end of the fusion protein contains 6 histidines of the vector. After the expression vector is transformed to the host bacteria, under induction of isopropyl-beta-D-thiogalactoside, the fusion protein and the TEV protease based on a chromosome are expressed at the same time; the TEV is acted on an enzyme cutting site between the maltose-binding protein and the target protein for separating the target protein. According to the intracellular shearing method, steps of fusion protein separating, TEV enzyme digesting, and the like are omitted.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for cleaving fusion proteins by protease in Escherichia coli cells. Background technique [0002] The high expression of heterologous genes in E. coli to obtain a large amount of protein is one of the most common forms of obtaining protein drugs, gene function research and protein research. Due to differences between species of organisms, many heterologous proteins exist in insoluble forms when expressed in E. coli. Insoluble proteins exist in inclusion bodies and need to be treated with a strong ionic denaturant to release the protein for purification, and then renature it with a reducing agent after purification. The process of denaturation and renaturation can easily lead to the reduction or loss of protein activity because of the need for strong oxidants and reducing agents, which will directly lead to the weakening of the therapeutic effect of protein drugs and t...

AI Technical Summary

Problems solved by technology

After the fusion protein is purified, the target protein is separated from the fusion tag by protease digestion to release the target protein, which includes two steps of protein purification and enzyme digestion. The experimental operation is difficult, and the yield of the target protein is often lower than Low; and because the purified protein is digested, the protein is easily inactive

In addition, highly specific proteases (such as TEV and PreScission) are very expensive, and large-scale use will inevitably cause a great economic burden, making it difficult to use them in the production of large-scale protein drugs

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