蛋白内含子介导纯化的表达载体的构建及应用  

Construction and Application of an Intein-mediated Prokaryotic Expression and Purification Vector

作　　者：张云龙[1] 张众[2] 王飞[2] 陈毓荃[1] 黄华樑[2] 

机构地区：[1]西北农林科技大学,陕西杨陵712100 [2]中国科学院遗传与发育研究所,北京100101

出　　处：《生物技术》2004年第4期13-16,共4页Biotechnology

摘　　要：将抗膀胱癌单链抗体 (PG)融合在蛋白内含子 (intein)的C末端 ,利用intein的N末端具有的chitinbindingdomain(CBD)对几丁质柱的吸附特性及intein的自切割活性对PG进行一次性纯化。同时 ,引入大肠杆菌分子伴侣肽基脯氨酰顺反异构酶 (Fk pA)与目的蛋白进行共表达及融合表达 ,以使目的蛋白以可溶形式存在。结果显示 ,PG融合蛋白对几丁质柱的吸附效率很高 ,in tein的自切割释放PG的效率也较高 ,但PG仅出现在SDS洗脱液中。FkpA与PG共表达能显著地提高目的蛋白的可溶性。Intein介导的纯化系统前景远大。When fused at intein' s C-terminus,single-chain Fv(scFv) against human bladder carcinoma can be purified in a single chromatographic step because chitin binding domain(CBD) fused at intein's N-terminus can bind to the chitin resin and intein possess a inducible self-cleavage activity to separate scFv from the affinity tag.In order to improve the soluable expression of target protein, scFv are coexpressed or fusion-expressed with E.coli peptidyl-prolyl cis,trans-isomerase FkpA.The results show that scFv can bind to chitin resin high efficiently and the self-cleavage efficiency of intein is also very high.scFv is only present in the SDS elution fraction.FkpA can be conducive to improve the soluable expression of scFv.Intein-mediated purification system have a bright future.

关 键 词：单链抗体 INTEIN CBD FkpA pTWIN 纯化 

分 类 号：R392.11[医药卫生—免疫学] Q789[医药卫生—基础医学]