Rosenfeld S A, Nadeau D, Tirado J, Hollis G F, Knabb R M, Jia S
Cardiovascular Sciences Department, DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880, USA.
Protein Expr Purif. 1996 Dec;8(4):476-82. doi: 10.1006/prep.1996.0127.
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.
重组水蛭素（HIR）是一种从医用水蛭 Hirudo medicinalis 中提取的强效凝血酶抑制剂，已在甲基营养型酵母毕赤酵母中克隆并表达。将 HIR 基因插入毕赤酵母 pPic9K 表达载体，使该基因的表达受醇氧化酶（AOX1）启动子控制，且 HIR 编码序列与酿酒酵母前体 - 前α-交配因子信号序列融合。pPic9K 上还存在 Tn903Kan(r) 决定簇和 His4 + 基因，为筛选 HIR 基因的染色体整合体提供了一种方法。将 DNA 电穿孔导入毕赤酵母菌株 GS115（his - 4）后，回收 His + 转化子并接种在含有浓度递增的氨基糖苷类抗生素 G418 的培养基上。所得的 His + G418 抗性转化子在摇瓶中培养，并筛选那些向生长培养基中分泌重组水蛭素（rHIR）的转化子。保留表现出 rHIR 产生和分泌的克隆用于发酵研究，发现优化生长条件可显著提高 rHIR 的表达。保留用于进一步表征的一个克隆以 1.5 g/升的水平分泌 rHIR。使用简单的两步色谱法，rHIR 纯化至纯度 > 97%，回收率为 63%。纯化的 rHIR 具有预测的 N 端氨基酸序列，并表现出与在其他异源系统中产生的多种 HIR 同工型相同的凝血酶抑制动力学。基于这些数据，毕赤酵母为生产和纯化数克量的具有生物活性的 rHIR 以进行结构/功能分析提供了一个高效的系统。
