Secretory expression of human insulin precursor in Pichia pastoris using truncated α-factor leader sequence and a short C-peptide

Abstract

In the past ten years, diabetes prevalence has increased rapidly in low- and middle-income countries due to lifestyle changes. Increasing number of diabetic patients led to the escalation of recombinant insulin demand which was creating a large global insulin market. Pichia pastoris has appeared as an alternative host to produce recombinant proteins. It had excellent qualifications as an expression host in large-scale production of recombinant proteins for therapeutic use. We attempted in this study to express the insulin precursor (IP) in P. pastoris. We used a synthetic IP-encoding gene constructed in a frame with the truncated α-factor secretory signal and a short C-peptide (DGK) linked A- and B-chain of human insulin in a pD902 expression vector. Several zeocin resistant clones have been successfully obtained and verified using PCR with AOX1 specific primers for the integration of the expression cassette into P. pastoris genome and for the identification of Mut phenotypes. The secretion of IP by P. pastoris clone was confirmed by SDS-PAGE of the culture supernatant. It showed a single band of the secreted IP with a molecular mass above 6.5 kDa.

Keywords

• diabetes, recombinant human insulin precursor, P. pastoris, α-factor and C-peptide.