Soluble Overexpression And Purification Of Bioactive Human Proinsulin In Escherichia Coli

Insulin promotes fat,glycogen,protein synthesis,reduce the level of blood sugar,it is one of the main drugs used to treat diabetes.Recombinant insulin expressed in E.coli as inclusion body,to get a complete insulin involves the denaturation and refolding,would greatly reduce the final yield of insulin.This article aims to explore achieve soluble expression of recombinant human proinsulin in E.coli by different fusion proteins.The main contents and results are as follows:Proinsulin gene was designed and synthesized.The DNA fragments were cloned into expression vector pHis-NusA?pHisTrx and pGEX-4T-2,and named as pHis-NusA-insulin?pHisTrx-insulin?pGEX-4T-2-insulin.Expressed in E.coli,the protein His-NusA-insulin?HisTrx-insulin all existed in the form of soluble body.Induction temperature?the final concentration of IPTG and induction time of His-NusA-insulin and HisTrx-insulin was optimized.Recombinant protein His-NusA-insulin expression was induced at 37 C 5 h,0.1mM of IPTG.Recombinant protein HisTrx-insulin expression conditions was 250C induced 5 h,0.1 mM of IPTG.Recombinant protein His-NusA-insulin and HisTrx-insulin was isolated and purified by affinity chromatography(Ni-beads).150 mM imidazole eluted impurities and 500 mM imidazole eluted His-NusA-insulin.Protein concentration of recombinant protein His-NusA-insulin was 1.059 ?g/?L measured by BradFord.100mM imidazole eluted impurities and 500 mM imidazole eluted HisTrx-insulin.Protein concentration of recombinant protein His-NusA-insulin was 0.491 ?g/?L measured by BradFord.Using different concentrations of TEV protease cleavage recombinant protein His-NusA-insulin,proinsulin detected.The experiment based on recombinant human proinsulin for the study,exploration and research by co-expression of the fusion protein to increase expression of recombinant soluble proinsulin,lay the foundation for reducing the production cost of recombinant insulin.