Production and purification of recombinant hirudin expressed in the methylotrophic yeast Pichia pastoris

Protein Expression and Purification
S A RosenfeldS Jia

Abstract

A recombinant form of hirudin (HIR), a potent thrombin inhibitor derived from the leech Hirudo medicinalis, was cloned and expressed in the methylotrophic yeast Pichia pastoris. The HIR gene was inserted into the P. pastoris pPic9K expression vector such that the gene's expression is under alcohol oxidase (AOX1) promoter control and the HIR coding sequence is fused to the Saccharomyces cerevisiae pre-pro alpha-mating factor signal sequence. A Tn903Kan(r) determinant and His4+ gene are also present on pPic9K, affording a method for selecting chromosomal integrants of the HIR gene. Following electroporation of the DNA into the P. pastoris strain GS115 (his-4), His+ transformants were recovered and plated on medium containing increasing concentrations of the aminoglycoside antibiotic G418. The resulting His+ G418-resistant transformants were grown in shake flasks and screened for those that secreted recombinant hirudin (rHIR) to the growth medium. Clones exhibiting rHIR production and secretion were retained for fermentation studies where optimization of growth conditions was found to dramatically increase rHIR expression. One clone that was retained for further characterization secreted rHIR at a level of 1.5 g/liter. Using a str...Continue Reading

Citations

Mar 27, 1999·Protein Expression and Purification·L BriandJ C Pernollet
Oct 6, 2004·Preparative Biochemistry & Biotechnology·Wei-Bin Zhou, Yuan-Xing Zhang
Mar 30, 2017·Journal of Industrial Microbiology & Biotechnology·Xiao-Dong WangYan Xu
Aug 11, 2018·Biotechnology & Genetic Engineering Reviews·Jianguo Zhang, Nana Lan
Mar 10, 2006·Applied Microbiology and Biotechnology·Anfeng XiaoYuanxing Zhang
Aug 1, 2012·Journal of Industrial Microbiology & Biotechnology·Cuicui HuangShuhua Tan
Jul 17, 2012·Bioorganicheskaia khimiia·M A KostrominaA I Miroshnikov
May 1, 2020·Biomedicines·Sarah Lemke, Andreas Vilcinskas
Jan 21, 2000·FEMS Microbiology Reviews·J L Cereghino, J M Cregg
May 1, 2021·3 Biotech·Pavel KhvatkovSergey Dolgov
May 4, 2021·Frontiers in Pharmacology·Chen JunrenPeng Cheng

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