ApxⅢ包涵体蛋白纯化和变性条件的研究

摘要

为摸索重组ApxⅢ蛋白的纯化条件，采用IPTG诱导pET～apxⅢA／JM109，表达产物以包涵体形式存在．收集诱导后菌体，经冻融-超声破菌得到粗包涵体，经10 mmol／L Tris—HCl、TritonX～100和2mol／L尿素依次洗涤．包涵体沉淀分别用含有8mol/L尿素和6mol／L盐酸胍的溶液变性溶解．聚丙酰胺凝胶电泳（SDS—PAGE）分析结果表明：经洗涤包涵体纯度提高；8mol／L尿素和6mol／L盐酸胍都不能使ApxⅢ包涵体完全溶解．%Recombinant ApxⅢ was expressed in E. coli under IPTG induction. It existed as inclusion body. Cells were harvested, freeze - thawed, sonicated and centrifuged after expression, and the pellet （crude ApxⅢ inclusion body） was washed by successive steps as follows： 10 mmol/LTris- HC1, 1% TritonX- 100 and 2mol/L Urea. The precipitate was resolved and denatured by 8 mol/L Urea and 6 mol/ L Guanidine Hydrochloride respectively. The results of SAS - PAGE analysis show that purity of ApxⅢ inclusion body is improved by washed. The ApxⅢ inclusion body could not be dissolved completely in 8mol/L Urea and 6mol/L Guanidine Hydrochloride.