International Journal on Advanced Science, Engineering and Information Technology, Vol. 12 (2022) No. 3, pages: 1001-1007, DOI:10.18517/ijaseit.12.3.15109

Evaluating Pulses and Modified Fed-batch Feeding of Methanol to Increase Expression Level of Human Insulin Precursor in Pichia Pastoris High-Density Cultivation

Eko Wahyu Putro, Dini Nurdiani, - Hariyatun, Nuruliawaty Utami, Wien Kusharyoto, Ario Betha Juanssilfero, Rozy Ayu Silvia

Abstract

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.

Keywords:

Diabetes; human insulin precursor; Pichia pastoris; bioreactor; fed-batch.

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