重组虎纹捕鸟蛛毒素Ⅰ用pET_(31)b载体的表达和纯化  

Expression of Recombinant HWTX-Ⅰ Gene by pET_(31)b Vector and Purification of Product

作　　者：刁建波[1] 刘君梁[2] 梁宋平[2] 

机构地区：[1]北京大学蛋白质工程及植物基因工程国家重点实验室,北京100871 [2]湖南师范大学生命科学学院,长沙410081

出　　处：《中国生物化学与分子生物学报》2003年第2期195-200,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金资助项目 (No .39990 60 0 )~~

摘　　要：化学全合成虎纹捕鸟蛛毒素 Ⅰ (HWTX Ⅰ )基因在大肠杆菌中被表达 ,采用pET31b载体 ,表达产物N端为大肠杆菌酮基立体异构酶的融合蛋白 ,经亲和连续 6个组氨酸的亲和柱层析后 ,溴化氰切割融合蛋白 ,再经高效液相色谱反相柱纯化 ,得到了重组虎纹捕鸟蛛毒素Ⅰ .质谱分析及N端测序表明 ,rHWTX Ⅰ系正确表达产物 .还原复性的rHWTX Ⅰ表现出与天然HWTXThe gene of spider toxin, HWTX-Ⅰ has been synthesized by chemical method and cloned into the prokaryotic vector pET 31b. The fusion protein, whose N terminus encodes the E.coli ketosteroid isomerase, was expressed by IPTG induction. After purification by His bind resin, the fusion protein was cleaved with CNBr and the recombinant HWTX-Ⅰ was purified by reversed phase HPLC. The purified rHWTX-Ⅰ was proved to be correctly expressed by mass spectrum and N-terminal sequencing. After denaturation and renaturation, the recombinant HWTX-Ⅰ was proved to have the same activity as that of the native one.

关 键 词：虎纹捕鸟蛛毒素Ⅰ 基因工程 基因克隆 基因表达 融合蛋白 纯化 pET31b载体 生物学活性 

分 类 号：R996.3[医药卫生—毒理学] Q78[医药卫生—药学]