Secretory Expression Of "Mini C" Human Proinsulin In The Methylotrophic Yeast Pichia Pastoris

As a drug for treating diabetes, human insulin has been expressed in E. coli and the yeast S. cerevisia, but for the shortage of themselves, the level of the expression is not high. The methylotropic yeast Pichia pastoris expression system has been developed recently, it has unparalleled superiority in expression heterologous proteins. Proinsulin gene has been expressed in Pichia pastoris successfully. People pay more attention to it because of the high expression level and post-translation modification of secreted proteins.The fused "Mini-C" human proinsulin analog (B-Arg-Arg-Lys-Arg-A) was inserted into the plasmid pPIC9 of Pichia pastoris to obtain secretory plasmid I-pPIC9. Electroporation of Pichia pastoris GS115 (his4), and pick up some high-copy transformants . After 1L shake flask, SDS-PAGE analysis indicated the efficient expression and secretion of recombinant human insulin, the expression level is 50mg/ml. After fermen, isolation with (NH₄)₂SO₄ and DEAE-Sephadex A-25 Ion-Exchange Chromatography, the the expression level of recombinant human insulin increased from 15% to 30%.Radiation Immunity Analysis of Insulin proved that recombinant human insulin have the same receptor binding capacity with humaninsulin; HPLC proved that recombinant human insulin have the same rettime with human insulin; Biological activity experiment in mouse proved that recombinant human insulin have the same in vivo biological activity with human insulin.