The Expression Of Human Insulin Precursor In Pichia Pastoris And Escherichia Coli

Insulin is not only the unique hormone that can lower blood sugar, but also promot to synthesis ofglycogen, fat and protein in human body. The receptor tyrosine kinase is the basis of mechanism. Proinsulin isconsists of three peptide chains of BCA, the insulin is a hypoglycemic activity of low molecule for removingthe C-peptide.On the basis of the human proinsulin IS structure, this reserch successfully constructed the recombinatsteains by using preferred encodes reverse encoding IS1gene, IS2gene and Pichia pastoris and Escherichiacoli expression systems. IS1was secreted in the form of constitutive expression in Pichia pastoris. Afterpurfying and removing C-pettide of proinsulin, the recombinat insulin has hypoglycemic activity in modelmouse. The fusin protein of proinsulin with self-splicing tag is high level expressed in the form of inclusionbodies in E. coli.IS1gene sequence was encoded using P. pastoris preferred codons and synthesized with restrictionenzyme sites XhoⅠand XbaⅠby the Shanghai Sangon Biotechnology Company，based on which recombinantplasmid pGAPZaA-IS1was constructed and transformed to P. pastoris SMD1168. The expressed protein wasidentified by Tricine-SDS-AGE and Western blot，then purified by column chromatography and ultrafiltration，and determined for hypoglycemic activity after removal of synthetic C peptide. Restriction analysis andsequencing proved that recombinant plasmid pGAPZaA-IS was constructed correctly. Tricine-SDS-PAGEshowed that the expressed recombinant protein was secreted into medium supernatant，with a relativemolecular mass of about6,000. After removal of C peptide，the purified recombinant protein showedhypoglycemic activity in model mouse.IS2gene sequence was encoded using E. coli preferred codons and synthesized with restriction enzymesites NruⅠ a ndEcoRⅠ b y the Shanghai Sangon Biotechnology Company. Rcombinant plasmid pTWIN1-IS2was constructed and transformed to E. coli BL21. The fusin protein of proinsulin with DBna self-splicing CBDtag is high level expressed by IPTG induced, was inclusion bodies in E. coli. Effect of temperature on theinclusion protein of soluble is small. Due to the complex process of refolding, we abandoned the method ofexpression proinsulin in E. coli.In summary, we successfully designed and constitutively expressed proinsulin by in Pichia pastoris. Theinterest protein has great hypoglycemic activity in mous by purification and removing C-peptides by trypsin.The results laid foundation for its further research.