Influence Of Expression Recombinant Human Insulin Precursor In High-cell Density Cultures Of Pichia Pastoris With Different Promoters

Insulin was the first recombinant product approved by the FDA for humanapplication. The first expression systems were based on the separate expression of the A-and B-chains fused to carrier proteins in two different E. coli strains. Nowadays, humaninsulin is produced as recombinant protein, using two major routes. One route involves theproduction of an insulin precursor in the form of inclusion bodies, using E. coli asexpression host with subsequent solubilization and refolding procedures. The other routeinvolves the utilization of yeastbased expression systems, leading to the secretion of asoluble insulin precursor into the culture supernatant. Due to the extensive experience inlarge-scale cultivation, Saccharomyces cerevisiae became the first yeast-based expressionsystem for secretory IP production. Though S. cerevisiae is still the predominant yeastsystem for insulin production, several alternative yeast hosts have become available inrecent years. Of these, the methylotrophic yeast Pichia pastoris has emerged as a veryuseful expression host with superior features. In particular, its strong and tightly regulatedmethanol-inducible alcohol oxidase1（AOX1） promoter and its capacity to reach very highcell densities by simple cultivation strategies collectively contribute to stable and highlevel production of recombinant proteins.Objectives: To construct the P. pastoris expression system of HumanMini-proinsulin DesB³⁰（the C peptide is AAK）,pPIC9K-HMPIDesB³⁰/GS115、pGAPZαA-HMPIDesB³⁰/GS115、 pPIC9K-pGAPZαA-HMPIDesB³⁰/GS115。Shake flaskexpression insulin precursor and choose the high level expression strain of FJ-H₁、FJ-H₂、FJ-H₃.To study the influence of Secretory expression recombinant human insulinprecursor in Pichia pastoris with different promoters.Methods:To construct the recombinant plasmid pGAPZαA-HMPIDesB³⁰andpPIC9K-HMPIDesB³⁰. The plasmid of pGAPZ α A-HMPIDesB³⁰and pPIC9K-HMPIDesB³⁰had been linearized with AvrII and SacⅠrespectively and integrated intothe P. pastoris strain of GS115’s genome by electrotransformation. The pGAPZαA-HMPIDesB³⁰/GS115and pPIC9K-HMPIDesB³⁰/GS115strain with multiple clones wereobtained by YPD plates which contains increasing concentrations of G418or Zeocin.The plasmid of pGAPZαA-HMPIDesB³⁰had been linearized with AvrII and integratedinto the P. pastoris strain of pPIC9K-HMPIDesB³⁰/GS115, to obtain pPIC9K-pGAPZα A-HMPIDesB³⁰/GS115strain. Expression of HMPIDesB³⁰and choose the high levelexpression strain of FJ-H₁(pPIC9K-HMPIDesB³⁰/GS115)、FJ-H₂(pGAPZαA-HMPIDesB³⁰/GS115)、FJ-H₃(pPIC9K-pGAPZαA-HMPIDesB³⁰/GS115).Optimizethe fermentation condition and ferment with high density.Results:The expression level of proinsulin in FJ-H₃strain fermented in30Lfermenter reached1.6g/L, which was1.6times higher than that in FJ-H₁（1.0g/L）and5.3times higher than that in FJ-H₂strain （0.3g/L）. combined use of GAP and AOX1promoter to enhance the expression of human insulin precursor in pichia pastoris. Thepurified protein was characterized by12%Tricine SDS-PAGE、 mass spectrometry andpeptide mapping, and the molecular mass of the expressed protein is in accordance withthe cumulated value. HMPIDesB³⁰can be secretorily expressed in pPIC9K-pGAPZαA-HMPIDesB³⁰／GS115strain.Clusions: HMPIDesB³⁰had been secretorily expressed in P. pastrois successfully,study the influence of Secretory expression recombinant human insulin precursor inPichia pastoris with different promoters. Combined use of GAP and AOX1promoter toenhance the expression of human insulin precursor in pichia pastoris.and The expressionlevel of proinsulin in FJ-H₃strain reached1.6g/L.The recombinant protein ofHMPIDesB³⁰had satisfactory biological activity, and it neither had glycosylationmodification nor formed dimer or multimer. The optimal conditions of the fermentationwere established. purificationan to boost the recovery rate.