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

M. Anggiani, I. Helianti, Abinawanto

Research output: Chapter in Book/Report/Conference proceedingConference contributionpeer-review

3 Citations (Scopus)

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.

Original languageEnglish
Title of host publicationProceedings of the 3rd International Symposium on Current Progress in Mathematics and Sciences 2017, ISCPMS 2017
EditorsRatna Yuniati, Terry Mart, Ivandini T. Anggraningrum, Djoko Triyono, Kiki A. Sugeng
PublisherAmerican Institute of Physics Inc.
ISBN (Electronic)9780735417410
DOIs
Publication statusPublished - 22 Oct 2018
Event3rd International Symposium on Current Progress in Mathematics and Sciences 2017, ISCPMS 2017 - Bali, Indonesia
Duration: 26 Jul 201727 Jul 2017

Publication series

NameAIP Conference Proceedings
Volume2023
ISSN (Print)0094-243X
ISSN (Electronic)1551-7616

Conference

Conference3rd International Symposium on Current Progress in Mathematics and Sciences 2017, ISCPMS 2017
Country/TerritoryIndonesia
CityBali
Period26/07/1727/07/17

Keywords

  • AOXI promoter
  • Pichia pastoris
  • Thermomyces lanuginosus lipase
  • synthetic gene

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