蛋白内含子介导纯化的表达载体的构建及应用

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chitin binding domain(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(FkpA)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，intein的自切割释放PG的效率也较高，但PG仅出现在SDS洗脱液中。FM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。

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

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.