人重组胱天蛋白酶原-3的表达和体外活化  

Over-expression and autoactivation of recombinant human procaspase-3

作　　者：李现亭[1] 夏东岚[1] 莫晓宁[1] 宋泉声[1] 张颖妹[1] 马大龙[1] 

机构地区：[1]北京大学基础医学院免疫学系,北京100083

出　　处：《北京大学学报（医学版）》2002年第4期316-320,共5页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金 ( 39870 42 7)资助项目~~

摘　　要：目的 :建立一个在短时间内获取大量活化胱天蛋白酶 3的方法。方法 :将胱天蛋白酶 3酶原的基因从真核表达载体中克隆到原核表达载体 pMTY4 His中 ,在大肠杆菌中以包涵体的形式表达MS2 胱天蛋白酶原 3融合蛋白 ;将包涵体变性 ,经酸化和浓缩进行体外活化 ;对所得蛋白用Westernblot和活性试验进行鉴定。结果 :构建成pMTY4 His 胱天蛋白酶 3原核表达载体 ,在大肠杆菌中获得了较高水平表达的胱天蛋白酶原 3酶原 MS2融合蛋白。体外活化后每升诱导菌可获得约 10mg蛋白 ,用抗胱天蛋白酶 3单克隆抗体证实所得蛋白为胱天蛋白酶原 3,活性试验证实得到高活性的蛋白。结论 :成功地建立了大量获取高活性的胱天蛋白酶 3的方法 ,为深入研究胱天蛋白酶Objective:To provide reliable and effective method of getting activated human caspase-3. Methods: The cDNA of procaspase-3 was subcloned into E coli expression vector pMTY4-His. MS2-procaspase-3 fusion protein was expressed in E coli as inclusion bodies (IB). The IB were solubilized in guanidine hydrochloride and dialyzed against 50% acetic acid overnight and diluted with 9 volumes of water. The mixture was diluted rapidly to one containing 1.0 mol·L -1 Tris, pH 8.0 and 5 mmol·L -1 DTT and then concentrated to a nearly-dry state on the ultrafiltration membrane, followed by dissolving the concentrated protein. Results: High level expression of MS2-procaspase-3 recombinant protein was achieved in E coli as IB. About 10 mg activated caspase-3 could be obtained from 1L cultured bacteria. The activated caspase-3 could efficiently cleave Ac-DEVD-AMC and was inhibited by Ac-DEVD-CHO. Conclusion: We have successfully established a procedure for obtaining large quantity of activated caspase-3,which could be used for studying the function and structure of caspase-3.

关 键 词：胱天蛋白酶原-3 体外活化 基因重组 细胞凋亡 单克隆抗体 

分 类 号：TQ464.8[化学工程—制药化工]