毕赤酵母高密度发酵表达血管紧张素转化酶C-结构域  

Optimization of Fermentation for ACE C-Domain from Pichia pastoris

作　　者：徐珏[1] 许传莲[1] 杜方尧[1] 

机构地区：[1]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

出　　处：《中国生物工程杂志》2010年第4期33-38,共6页China Biotechnology

基　　金：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

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5L发酵罐中,对重组毕赤酵母表达ACEC-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,ACEC-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,ACEC-结构域表达量和酶活力分别达到446mg/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ACEC-结构域蛋白,筛选专一性更强的ACEC-结构域抑制剂奠定了基础。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.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 concentrations 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.

关 键 词：毕赤酵母 ACE C-结构域 蛋白降解 发酵 

分 类 号：Q516[生物学—生物化学]