| Human insulin is an indispensable medicine for patients with IDDM (insulin-dependent diabetes mellitus)in spite of there are several compounds with some blood sugar-lowering effect. Therefore, high expression of the recombinant human insulin is a great target ,which has attracted more and more attention from the researchers around the world. The methylotrophic yeast Pichia pastoris is a recently developed system for expressing heterologous proteins. Our purpose is to highly express the human insulin by this system.Firstly, we selected the secreting plasmid pPIC9k as vector to construct recombinant plasmid pPIC9k/HIP and yeast Pichia pastoris GS115 as the gene-expression host.After physical mapping and the nucleotide sequencing of the purpose gene, the recombinant plasmid pPIC9k/HIP was linearnized by restriction endonuclease BglII and transformed into the host GS115 by electroporation. The G418 (geneticin) resistance was used for screening high-copy transformants from the gained his+Muts phenotype 86 transformants. After fermentation of the chosen strains,the fermentation supernate was determined by Tricine-SDS-PAGE,which indicated the transgenic yeast strains could highly express and secrete the HIP. Moreover, it proved that the expression level increased with the increasing gene copy in recombinants. The expression quantity of one high-copy strain indicated the expressing pro-insulin increased with time course and reached the maximum expression after 72 hours induced. By ultrafiltration and other methods we got a little of crude HIP, which was further processed with CNBr,Carboxypeptidase A and Carboxypeptidase B to produce mature insulin. Immunoassay proved that this sample has bioactivity liking the natural human insulin.In short, we constructed recombinant HIP Pichia pastoris strains, which can express HIP successfully. The bioactivity of our sample had been comfirmed by immunoassay. Thus, the strains and the sample processing is useful for our further study.
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