Application of urokinase receptor stable mutant suPARcc in eukaryotic extracellular protein expression

Abstract

The invention belongs to the field of biotechnology, and relates to an application of a soluble urokinase receptor mutant suPARH47C/N259C (suPARcc) in eukaryotic extracellular protein expression, specifically increasing expression level of exogenous protein by means of fusion expression, and further utilizing high affinity of suPARcc to a ligand ATF, and utilizing an ATF affinity column to directly and efficiently capture a target protein from a large-volume fermentation broth. According to the application, suPARcc and a conventional protein enzyme digestion site are integrated into a eukaryotic expression system vector to obtain a suPARcc gene fusion expression vector, a target gene to be expressed and the suPARcc gene are fused in a correct reading frame to construct a fusion expressionrecombinant plasmid, the fusion expression recombinant plasmid is transfected and screened for stable cell lines, a fusion protein containing the tag and the target protein is obtained through induction, secretion and expression in eukaryotic cells, and the fusion tag can be finally removed by a conventional protease.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the application of suPARcc, a stable mutant of urokinase receptor, in the expression of eukaryotic extracellular protein. Background technique [0002] With the development of genetic engineering technology, genomics and proteology technology, recombinant protein has been widely used in the fields of biology, medicine, agriculture and animal husbandry and veterinary medicine. However, the acquisition of large-scale biologically active eukaryotic proteins is still a difficult point in recombinant protein technology. There are two types of expression systems for recombinant proteins: prokaryotic and eukaryotic. The prokaryotic expression system represented by Escherichia coli has the advantages of high expression, easy operation and low production cost, but it is often impossible to obtain biologically active proteins when expressing eukaryotic proteins that require complex post-translat...

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Problems solved by technology

However, the natural suPAR has a flexible structure and may interfere with the target protein, so it is not suitable for high-efficiency expression and purification of the target protein as a fusion protein tag. Therefore, we used a conformationally stable mutant of suPAR, suPAR H47C / N259C (suPARcc) to conduct this study

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