毕赤酵母高密度发酵表达血管紧张素转化酶C-结构域

中国生物工程杂志

2010, Vol. 30

Issue (04): 33-38

技术与方法

毕赤酵母高密度发酵表达血管紧张素转化酶C-结构域

徐珏,许传莲^**,杜方尧

浙江理工大学生命科学学院蛋白质组与分子酶学实验室杭州 310018

Optimization of Fermentation for ACE C- Domain from Pichia pastoris

XU Jue,XU Chuan-lian,DU Fang-yao

全文: PDF(774 KB) HTML

摘要：

血管紧张素转化酶（ACE, EC3.4.15.1）在调节血压方面具有重要作用。研究证实，ACE的C结构域（ACE-C）是使血管紧张素I (AngI)分解的主要活性位点。在5 L 发酵罐中, 对重组毕赤酵母表达ACE C-结构域的发酵工艺进行优化，探讨温度、pH、甲醇浓度等主要因素对重组蛋白表达量和酶活力的影响。结果表明,当工业培养基添加2%蛋白胨为氮源时, ACE C-结构域的降解现象得到了有效控制;采用诱导温度为26℃,pH5.5,甲醇含量为1.5%的表达条件，ACE C-结构域表达量和酶活力分别达到446 mg/L和38.2U/ml，比活力达到86U/mg，是Sigma公司ACE标准品比活力的2倍，为大规模制备ACE C-结构域蛋白，筛选专一性更强的ACE C-结构域抑制剂奠定了基础。

关键词： 毕赤酵母; ACE C-结构域; 蛋白降解; 发酵

Abstract:

Angiotensin I-converting enzyme (ACE, EC3.4.15.1) plays an important role in regulating blood pressure. Now, ACE C-domain is identified to be the main site of angiotensin I cleavage in Vivo. In this study, the high expression recombinant Pichia pastoris was constructed and the screening tests was performed in 5 L bio- reactor to obtain the optimal values of several key fermentation parameters. Based on effects on the expression level, the optimal values for the temperature, the con- centrations of methanol and the pH were 26℃, 1.5% (V/V) and 5.5, respectively. Addition of 2%polypeptone to substrate would effectively repress proteolysis. The application of these optimal parameters successfully achieved high-throughput production: the cell density (OD600) of recombinant Pichia pastoris and the yield of crude target protein were respectively 397 mg/L and 446 mg/ L. After the purification with Ni-NTA columns, ACE C-domain was collected with a purity of 98.6%, and the specific activity of it was reached 86 U/mg, which is double fold than that of ACE purchased from Sigma. This provided a zymolytic condition to be used for ACE C-domain in industrial scale production, and provided a high specific activity enzyme for screening specific inhibitor to ACE C-domain.

Key words: Pichia pastoris ACE C-domain Proteolysis Fermentation

收稿日期: 2010-01-08 出版日期: 2010-04-29

基金资助:

浙江省教育厅重点项目（Z200804057）、浙江省大学生科技创新推广项目(14080131380912)资助项目

通讯作者: 许传莲 E-mail: chuanlianxu@163.com

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引用本文:

徐珏许传莲杜方尧. 毕赤酵母高密度发酵表达血管紧张素转化酶C-结构域[J]. 中国生物工程杂志, 2010, 30(04): 33-38.

XU Jue, HU Chuan-Lian, DU Fang-Yao. Optimization of Fermentation for ACE C- Domain from Pichia pastoris. China Biotechnology, 2010, 30(04): 33-38.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I04/33

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