Expression, Processing And Bioactivity Analysis Of HGH, HSA-double Arg C Peptide Recombinant Insulin

Insulin is a kind of essential medicine in the clinical treatment of patients with severe type 1 and type 2 diabetes.As the dramatic increase in diabetes, the insulin market demand is increasing rapidly. Thus,the development of low cost, high activity and low side effect recombinant human insulin raw materials has become a hot point in the world.At present, the way to enhance the expression of recombinant human insulin is mainly by optimizing the fermentation technology. However, it existed neight expression limitation nor the renaturation level is relatively low. As the molecular weight of insulin is relatively small, individual expression at the transcription,translation level is unstable which is vulnerable to degradate.In this study,we used genetic engineering technique to achieve human proinsulin (containing only two amino acids for the C peptide) fusion protein with the leader sequence at N-terminal side, improving the expression of genetic recombinant insulin in E. coli by optimization the conditions at all aspects,Improving the stability and expression level of inclusion body protein which lay the basis for the development of low cost and high activity of genetically engineered human insulin raw materials.The results are summarized as follows:Two precursor sequences choosed as a precursor peptide in this experiment were available:hGH,N-terminal section of human growth hormone and HSA,N-terminal section of human serum albumin, meanwhile,the C peptide is designed for two arginines,synthesizing 10-long oligonucleotide chains to amplify hGH-double Arg C peptide human proinsulin gene by overlap extension PCR technique (SOE PCR); at the same time,taking HSA-PUC19 plasmid kept in our laboratory as template to get HSA-double Arg C peptide human proinsulin gene. Then the two genes were inserted into PET-30a expression vector before transformed into Ecoli BL21 (DE3) strain.The high efficiency expression vector expressed in E.coli BL21(DE3) was successfully induced by IPTG in the form of insoluble inclusion bodies, accounting for about 30% of total proteins in E.coli.However,we do not observe the specific band expressed by HSA-double Arg C peptide human proinsulin gene.Then the expressed fusion protein was purified by Ni-NTA Affinity chromatography,After Renaturation, Freeze-dried procedures.The purified protein was digested by trypsinase and carboxypeptidase B,at last the digestion products were under DEAE Sepharose Fast Flow purification procedure in order to get the purified human insulin. After these processes we got a single band protein detected by SDS-PAGE analysis. Western BlotAnalysius showed that the recombiant protein possessed Insulin antigenicity, the subcutaneous injection of mice showed the insulin have a significant ability to lower blood glucose.Conclusion:The precursor sequence combined with c peptide was highly expressed in E.coli succefully,establishing an efficient method for recombinant human insulin production,which also lay the foundation for precursor sequence mutation in order to promote the folding efficiency of recombinant insulin based on the precursor peptide.