BACE蛋白的表达、纯化和活性测定  

Expression, Purification and Activity Measurement of Soluble BACE Protein in E.coli

作　　者：魏晓超[1] 李恒[1] 纪建国[1] 李茹 任燕飞 冯金辉 郝福英[1] 

机构地区：[1]北京大学生命科学学院生物大分子实验室,北京100871

出　　处：《生物化学与生物物理学报》2002年第4期498-501,共4页

基　　金：教育部留学回国人员科研启动基金资助项目 (No .940 6)~~

摘　　要：在大肠杆菌中表达、纯化并重新折叠以获得有活性的酸性蛋白水解酶 (BACE蛋白 )———一种与阿尔茨海默病 (AD)发病相关的蛋白水解酶。克隆BACE活性区的表达序列到原核表达载体 pET11a中 ,经E .coliBL2 1(DE3)表达 ,从包涵体中获取蛋白质 ,电泳鉴定后经梯度反向快速折叠法重新折叠 ,柱层析分离纯化 ,得到了表达的重组可溶性BACE蛋白 ;用高效液相色谱、质谱等方法检测其对人工合成多肽底物的水解作用 ;测定了BACE蛋白的酶促动力学常数。结果表明 ,得到的重组BACE蛋白具有水解人工合成小肽底物的活性。To express BACE (beta site APP cleaving enzyme), an aspartic protease related to Alzheimer's disease in E.coli to obtain the active protein after refolding and purification, the sequence for soluble form of BACE was inserted into prokaryotic expression vector pET11a. After expression in E.coli BL21(DE3), the protein was obtained as inclusion bodies, after refolding and purification. The proteolytic activity of the protein was measured by HPLC and MS. After refolding and purification, the protein exhibited beta secretase activity by cleaving the synthetic peptide, which was designed according to the beta secretase site at Swedish mutant of APP, and according Hanes method the kinetics constants for the synthetic peptide of BACE protein were measured. The study will facilitate BACE protein structure determination and inhibitor development efforts, which may lead to the evolution of promising and effective treatments for Alzheimer's disease.

关 键 词：BACE蛋白 表达 纯化 活性测定 阿尔茨海默病 Β分泌酶 重新折叠 

分 类 号：Q786[生物学—分子生物学]