【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。更多还原

【Abstract】 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.更多还原