title: Production and purification of recombinant hirudin expressed in the methylotrophic yeast Pichia pastoris.
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 straightforward two-step chromatography procedure, the rHIR was purified to > 97% with a recovery yield of 63%. The purified rHIR had the predicted N-terminal amino acid sequence and exhibited the same thrombin inhibition kinetics as a variety of HIR isoforms produced in other heterologous systems. Based on these data, P. pastoris offers an efficient system for production and purification of multigram quantities of biologically active rHIR for structure/function analyses.
authors.affiliation: Cardiovascular Sciences Department, DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880, USA. Rosenfsa@a.1.lldmpc.umc.dupont.com
authors.forename: S A
authors.initials: SA
authors.lastname: Rosenfeld
authors.forename: D
authors.initials: D
authors.lastname: Nadeau
authors.forename: J
authors.initials: J
authors.lastname: Tirado
authors.forename: G F
authors.initials: GF
authors.lastname: Hollis
authors.forename: R M
authors.initials: RM
authors.lastname: Knabb
authors.forename: S
authors.initials: S
authors.lastname: Jia
mesh_terms: D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D005285:Fermentation; D005822:Genetic Vectors; D006629:Hirudins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010843:Pichia; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae
chemical_list: D006629:Hirudins; D011994:Recombinant Proteins