Highly secretory expression system for proinsulin gene:Methylotrophic yeast Pichia pastoris

To obtain the high-level expression of human insulin, the methylotropic yeast Pichia pastoris was used as expression system. Pichia pastoris uses methanol as a sole carbon source. Alcohol oxidase(AOX) promoter as one of the strongest promoters can regulate the expression of heterologous proteins. The system has many advantages:(1)the techniques needed for the molecular genetic manipulation of Pichia pastoris are simplier;(2)Pichia pastoris can produce foreign proteins at high levels, either intracellularly or extracellularly;(3)the proteins can be performed many eukaryotic posttranslational modifications. The fused mini-C or non-C human proinsulin analog was cloned onto yeast chromosome, and under induction by methanol, the expressed proinsulin was secreted into medium at the level of 250300 mg in 1 L fermentation culture. The expression level accounted for 20% of total bacterial proteins. The recombinant human insulin has the same receptor binding capacity and biology activity with pig insulin.