Transformation and characterization of human insulin precursor gene in Pichiapastoris X-33

The case of diabetes increases significantly and has been projected to reach 592 million people in 2035. Consequently, the necessity of insulin will rise manifold and an efficient production system for insulin production is required to meet the market demands. The human insulin precursors that enzymatically converted to human insulin can be produced using Escherichia coli, Saccharomyces cerevisiae, or Pichia pastoris. In this study, Pichia pastoris is used for production human insulin precursor because the resulting recombinant protein can be folded accordingly and secreted to the external environment of the cell that simplifies the purification process. The study was initiated with the insertion of a synthetic gene of human insulin precursor into the pPICZaA to create recombinant pPICZaA-IP plasmid. The recombinant plasmid was transformed into Escherichia coli Top10 which then isolated and digested by the SacI enzyme. The linearize pPICZaA-IP plasmid was transfected into Pichia pastoris X-33 by electroporator. The result of transformation process, a total of 20 colonies of P pastoris X-33 were selected and inoculated in YPD agar medium containing Zeocin. The two colonies of P pastoris were characterized by PCR and sequencing showed that the recombinant pPICZaA-IP plasmid was successfully integrated into selected colonies of P pastoris.

Enhancement in Human Insulin Precursor Secretion by Pichia pastoris through Modification of Expression Conditions

Pichia pastoris is an alternative yeast expression system to produce heterologous proteins. It has excellent characteristics for an industrial cell factory, such as its ability to reach high cell densities, high secretory capacity, and a low level of native proteins. In our previous study, we introduced a synthetic insulin precursor (IP)-encoding gene constructed in a pD902 expression vector into P. pastoris. However, the P. pastoris recombinant strains expressed a little amount of IP protein. Here, we modified the expression conditions, including inoculum density, methanol concentration, methanol induction time, pH, and temperature, to obtain a higher amount of secreted IP than our previous result. Protein analysis for studying the five parameters was conducted by SDS-PAGE, and the protein amount was estimated by ImageJ applying lysozyme as standard. We successfully enhanced the IP expression by modifying expression conditions. The highest increased of up to 100 folds was achieved when methanol concentration for induction was arranged at 3% (v/v), and the initial cell density for methanol induction was set at an optical density at 600 nm (OD600) of approximately 10 compared to the standard procedure, where the expression was set at 0.5% (v/v) methanol induction and initial cell density at OD600 = 1.

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

In the past ten years, diabetes prevalence has increased rapidly in low- and middle-income countries due to lifestyle changes. This increased number of diabetic patients leads to the escalation of recombinant insulin demand, which is creating a large global insulin market. Pichia pastoris has appeared as an alternative host to produce recombinant proteins. It has excellent qualifications as an expression host for large-scale production of recombinant proteins for therapeutic use. In this study, we attempted to express the insulin precursor (IP) in P. pastoris. We used a synthetic IP-encoding gene constructed in 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 were successfully obtained and verified with PCR using AOX1 specific primers for the integration of the expression cassette into the P. pastoris genome and for the identification of Mut phenotypes. The secretion of IP by the Pichia pastoris clone in the culture supernatant was confirmed using SDS-PAGE, where a single band of the secreted IP with a molecular mass above 6.5 kDa was found.