三种Bcl-2家族蛋白的表达、纯化及活性分析  

Expression,Purification and Activity Analyses of Three Bcl-2 Family Proteins

作　　者：朱翠侠[1] 李勋[1] 李雯雯[1] 石志敏[1] 周佳海[1] 王任小[1] 

机构地区：[1]中国科学院上海有机化学研究所生命有机化学国家重点实验室,上海200032

出　　处：《生物医学工程学杂志》2010年第4期834-841,共8页Journal of Biomedical Engineering

基　　金：国家自然科学基金资助项目(90813006;20621062);上海市科委资助项目(074319113)

摘　　要：Bcl-2家族蛋白(Bcl-xL、Bcl-2、Mcl-1等)是细胞凋亡、自吞噬等生命过程的重要调控因子,是近年来热门的抗肿瘤药物靶标。从文献报道的蛋白序列出发,克隆用于表达Bcl-2蛋白的人源Bcl-2/Bcl-xL嵌合基因以及用于表达Mcl-1蛋白的人源与鼠源Mcl-1嵌合基因,分别构建谷胱甘肽巯基转移酶(GST)和组氨酸标签融合基因的原核表达质粒,建立这些融合蛋白在大肠杆菌中的高效表达及纯化方法,以及GST融合的Bcl-xL蛋白的表达纯化方法。通过基于荧光偏振原理的实验方法,测定表达纯化得到的目标蛋白与Bid BH3多肽的结合常数,所得结果与文献报道值接近,而且带有GST标签的融合蛋白与无标签蛋白的活性基本一致。这些成功表达纯化的目标蛋白可应用于以Bcl-2家族蛋白为靶标的小分子调控剂的体外筛选。Bcl-2 family proteins（Bcl-xL,Bcl-2,Mcl-1 etc.） are key regulators of some life processes,including apoptosis and autophagy.They are currently considered as promising targets for developing new anti-tumor therapies.In our study,the human Bcl-2/Bcl-xL chimeric gene and the human/mouse Mcl-1 chimeric gene were designed and cloned,and the prokaryotic expression vectors for expressing glutathione S-transferase（GST） fusion proteins and histidine tag fusion proteins were constructed respectively.These two proteins as well as the GST-Bcl-xL fusion protein were all successfully expressed in E.coli and subsequently purified.In addition,we measured the binding of these Bcl-2 family proteins to the Bid BH3 peptide by fluorescence polarization-based assay.The dissociation constants（Kd） obtained by us were in general agreement with the data reported in literature.The Kd values of all three proteins with or without the GST tag were almost identical.All these results validate the biological functions of these Bcl-2 family proteins obtained by us.These proteins can be used in the experimental screening of small-molecule regulators of Bcl-2 family proteins in vitro.

关 键 词：BCL-2家族蛋白 表达 纯化 荧光偏振 体外筛选 

分 类 号：Q78[生物学—分子生物学]