Optimization of methanol induction for expression of synthetic gene Thermomyces lanuginosus lipase in Pichia pastoris

Abstract

Methylotrophic yeast is widely used as a recombinant protein expression system. As a methylotrophic yeast, Pichia pastoris is capable for metabolizing methanol as its sole carbon source. The strong promotor AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. The aim of this research was to determine the optimum concentration of methanol for the expression of the synthetic Thermomyces lanuginosus lipase gene. In this study, synthetic lipase gene from T. lanuginosus lipase was cloned into Pichia pastoris GS115 (mut+) with electroporation method and expressed under the control of AOX1 promotor. Furthermore, colonies that grew on yeast extract peptone dextrose (YPD) medium containing 100 µg.mL−1 zeocin were confirmed using specific primers. Positive transformants were used as the seed culture for cultivation production. The concentrations of methanol, which is used to induce the target gene product, are 0.5%, 1%, 1.5%, 2%, 2.5%, and 3%, respectively. Every 24 hours for 5 days methanol was added to the production culture. Crude enzyme was sampled every 24 hours to test the activity of lipase and protein expression was analyzed using SDS-PAGE. The result showed that the 35 kDa protein recombinant has been expressed. The optimum concentration of methanol was 3% with the activity of lipase reached 9.625 U.mL−1 on the 3rd day. This result will then be applied in production of recombinant gene product used for further characterization.