Effect of Methanol Induction and Incubation Time on Expression of Human Erythropoietin in Methylotropic Yeast Pichia Pastoris

Authors

Adi Santoso, Research Center for Biotechnology, Indonesian Institute of Science, Cibinong, Bogor 16911, IndonesiaFollow
Neng Herawati, Research Center for Biotechnology, Indonesian Institute of Science, Cibinong, Bogor 16911, Indonesia
Yana Rubiana, Research Center for Biotechnology, Indonesian Institute of Science, Cibinong, Bogor 16911, Indonesia

Abstract

Erythropoietin (EPO) is a glycoprotein hormone consists of 165 amino acids and has molecular mass of 30,400 Daltons. The large quantities of these hormone required to satisfy clinical demand are currently met by recombinant expression in mammalian cell, namely chinese hamster ovary (CHO). Pichia pastoris has become popular yeast based protein production systems to substitute mammalian expression systems. P. pastoris is capable to use methanol as sole carbon and energy source. In this study, recombinant human EPO (rhEPO) protein obtained by expressing the hEPO gene in methylotropic yeast P. pastoris, strain X33. The present work was carried out to study the optimal methanol concentration for induction and the incubation time to obtain rhEPO protein. To perform this study, the transformed P. pastoris was induced with various concentrations of methanol (0%, 0.5%, 1%, 2.5%, 5%, 10%, and 20%) and incubation times (0 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, and 144 hours). The results demonstrate that the highest protein expression level occurred at concentration of 2.5% methanol induction, while the optimal incubation time was at 48 hrs.

Bahasa Abstract

Pengaruh Induksi Methanol dan Waktu Inkubasi terhadap Ekspresi Eritropoietin Manusia pada Khamir Metilotropik Pichia pastoris. Eritropoietin (EPO) adalah hormon glikoprotein yang terdiri dari 165 asam amino dan memiliki berat molekul sebesar 30.400 Daltons. Sebagian besar kebutuhan EPO didapatkan dari hasil sintesis pada sel mamalia Chinese hamster ovary (CHO). Pichia pastoris adalah sejenis khamir yang populer digunakan untuk menggantikan sistem ekspresi pada sel mamalia. P. pastoris dapat menggunakan metanol sebagai satu-satunya sumber energi karbon. Pada studi ini, protein rekombinan EPO (rhEPO) disintesis dengan cara mengekspresikan gen hEPO pada khamir metilotropik P. pastoris strain X33. Studi ini dilakukan untuk mengetahui konsentrasi metanol dan waktu inkubasi yang optimal untuk mensintesis rhEPO. Pada studi ini konsentrasi metanol yang digunakan adalah 0%, 0.5%, 1%, 2.5%, 5%, 10%, dan 20%. Sedangkan waktu inkubasi yang digunakan adalah 0 jam, 24 jam, 48 jam, 72 jam, 96 jam, 120 jam, and 144 jam. Hasil penelitian ini menunjukkan bahwa ekspresi protein yang tertinggi terjadi pada konsentrasi metanol sebesar 2.5% dan waktu inkubasi selama 48 jam.

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Recommended Citation

Santoso, Adi; Herawati, Neng; and Rubiana, Yana (2012) "Effect of Methanol Induction and Incubation Time on Expression of Human Erythropoietin in Methylotropic Yeast Pichia Pastoris," Makara Journal of Technology: Vol. 16: Iss. 1, Article 5.
DOI: 10.7454/mst.v16i1.1041
Available at: https://scholarhub.ui.ac.id/mjt/vol16/iss1/5