人激肽释放酶-1在甲醇酵母中高水平表达纯化与鉴定.PDF

生 物 工 程 学 报 Chin J Biotech 2008, July 25; 24(7): 1186-1193 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ © 2008 Institute of Microbiology, CAS CSM, All rights reserved 研究报告 人激肽释放酶-1 在甲醇酵母中高水平表达、纯化与鉴定 黄秀东, 王书生, 陈佩新, 王俊, 陈耀国, 潘学工, 曹之舫 上海万兴生物制药有限公司新药开发部, 上海 201026 摘 要: 将自肾脏cDNA 中获得的人激肽释放酶-1(Human kallikrein 1, hK1)基因插入到pPICZαA 载体中, 构建甲醇酵母 分泌型表达载体pPICZα-hK1, 该载体转化毕赤酵母(Pichia pastoris)宿主菌X33, 通过提高YPD 平板和培养液中抗生素 Zeocin 的浓度(500~700 μg/mL), 筛选能高水平表达重组人激肽释放酶-1(Recombinant human kallikrein 1, rhK1) 的 P. pastoris 工程菌株。在30 L 发酵罐中发酵的表达条件为30oC 、pH 6.0, 诱导64 h 时, 发酵上清中rhK1 产量达到6500 u/L(约 1.25 g/L)。表达的rhK1 有N-型糖基化程度不同的两种类型, 分别是分子量偏大的rhK1-H 和分子量略小的rhK1-L 。通 过苯基疏水作用、Cu2+螯合以及阴离子交换层析纯化, 从每升发酵上清中可获得0.28 g 的rhK1-H 和0.62 g 的rhK1-L, 纯 化的总得率约为72%, 纯度不低于96% 。到目前为止, 该研究获得的rhK1 产量高于其他研究者的结果。 关键词: 激肽释放酶, 毕赤酵母, 重组蛋白, S-2266 生色底物 High Level Expression, Purification and Characterization of Human Kallikrein-1 in Pichia pastoris Xiudong Huang, Shusheng Wang, Peixin Chen, Jun Wang, Yaoguo Chen, Xuegong Pan, and Zhifang Cao Department of Research Development , Wangxing Biopharmaceuticals Co., Ltd, Shanghai 201026, China Abstract: Human kallikrein-1 (hK1) gene was cloned from kidney tissues cDNA, it was inserted into the plasmid pPICZαA, then the yeast expression vector pPICZα-hK1 was constructed. After transformed into Pichia pastoris host X33, high-level expression transformants were screened by escalating the concentration of Zeocin (from 500 to 700 μg/mL) of YPD plate and medium. When temperature was 30oC, pH 6.0 with induction duration