Study On The Expression And Activity Of A New Recombinant Human Insulin In E.coli

Objective:As the first FDA-approved protein drug,recombinant insulin is widely used in the treatment of both type 1 and 2 diabetes.Currently,subcutaneous injection is the only available route for administration of exogenous insulin.The main drawback of insulin injection is low compliance caused by the psychological stress of needle insertion and pain.Therefore,in the past twenty years significant efforts have been made to explore the possibility of delivering insulin via the oral route.Oral insulin delivery,though attractive compared to injections,is currently technically challenging due to poor epithelial permeability and rapid enzymatic degradation of the insulin molecule within the gastrointestinal tract.This study attempts to genetically modify the human proinsulin by inserting a binding peptide(HAIYPRH)and fusion with a Elastin-Like Polypeptide(ELP).The goal of these genetic modifications are two-fold:to facilitate rapid purification of recombinant insulin and to increase absorption of insulin by the intestine epithelial cells.Methods:An expression plasmid pET-ProINS(7pep)-LE-Elp was introduced into the E.coil strain BL21(DE3)through heat shock transformation.The ProINS(7pep)-LE-Elp was purified by elastin-Iike polypeptide(Elp)tag and functionally tested in cultured human hepatic cell line HepG2 and in a mouse model of diabetes.Results:The ProINS(7pep)-LE-Elp fusion protein exists mainly in the form of inclusion bodies in E.coli cells.SDS-PAGE analysis showed that ProINS(7pep)-LE-Elp fusion protein has a molecular mass of approximately 45 kDa.Following denaturating/renaturing protocol and inverse transition cycling,soluble protein ProINS(7pep)-LE-Elp with purity over 80%was obtained.When intraperitoneally injected into diabetic mice,ProINS(7pep)-LE-Elp showed glucose-lowering activity.ProINS(7pep)-LE-Elp fusion protein also showed activity in HepG2 cells,as evidenced by stimulated the activation of Akt and decreased expression of PEPCK.Surprisingly,however,when ProINS(7pep)-LE-Elp fusion protein was injected into the colon of mice,it showed no glucose-lowering activity,suggesting that this fusion protein is unable to be absorbed by intestinal epithelial cells.Conclusion:A novel and bioactive long-acting ProINS(7pep)-LE-Elp fusion protein was successfully expressed,which laid the foundation for the development of novel insulin analogues.