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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.

No abstract

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.

Production of sufficient insulin at a more affordable price is necessary. The increase in the number of people living with diabetes puts more burden on healthcare and the economy. P. pastoris is a promising host to produce human insulin precursors at a high yield in minimal medium and secretes low levels of endogenous protein impurities. Production of the precursor involves several parameters, including glycerol concentration, culture density, methanol concentration, and medium composition. This study evaluated the effect of those parameters on protein expression in the flask culture. Subsequently, fermentation in the bioreactor was carried on according to the information obtained from flask culture. Methanol feeding to induce protein expression was undertaken by pulses and fed-batch modes. The fed-batch method was modified from a standard technique by incorporating constant flow rates with variable feed concentrations. Cell density was determined based on optical density measurement at 600 nm and dry cell weight. Tricine SDS-PAGE and reversed-phase HPLC conducted protein analysis. The pulse feeding produced higher precursor concentrations at ~445 mg/L than modified fed-batch feeding at ~267 mg/L. However, the modified fed-batch feeding can be an alternative to producing human insulin precursors when a standard fed-batch feeding with variable flow rates and 100% (v/v) methanol feed is difficult to apply.