PKB的PH结构域在原核表达及其纯化和活性  

Expression of the Pleckstrin Homology Domain of the Protein Kinase B in Prokaryotic System and Its Purification and Biological Assay

作　　者：施冬云[1] 魏廉[1] 黄伟达[2] 刘珊林[1] 徐伯赢[1] 申宗侯[1] 

机构地区：[1]复旦大学医学院生化与分子生物学系,上海200032 [2]复旦大学生命科学学院生化系,上海200433

出　　处：《复旦学报（医学版）》2002年第6期423-427,共5页Fudan University Journal of Medical Sciences

基　　金：上海市教委重点科研项目 (B990 80 6 );上海市自然科学基金 ( 0 2ZB14 0 19)资助

摘　　要：目的　利用原核系统表达原癌基因丝苏氨酸蛋白激酶B(PKB)的PH结构域 ,为进一步研究其结构和功能奠定基础。方法　我们构建了PKB的PH结构域的表达质粒 ,在大肠杆菌中表达出PH结构域 ,并利用蛋白N端加入的组氨酸标签进行亲和层析及离子交换层析纯化蛋白。结果　在大肠杆菌中表达的PH结构域以包涵体形式存在 ,经变性和复性后成为具有天然构象的可溶性蛋白 ,经亲和层析和离子交换层析纯化的PH结构域具有与磷脂酰肌醇PI 4 ,5 P2 的结合能力 ,证明其具有生物学活性。结论　本方法成功表达纯化了PKB的PH结构域 ,可用作PH结构域结构和功能的进一步研究。Purpose: To express the PH domain of the protooncogenic serine/threonine protein kinase B (PKB) in order to further study for its structure and function. Methods: A PKB PH domain prokaryotic expression vector was constructed and expressed in Escherichia coli. Purification was achieved first by a Ni (II) affinity column by virtue of a(His)10 tag on the protein, and then on a anion-exchange column. Results: The expressed PH domain in Escherichia coli was formed in inclusion body. After denaturation and renaturation, the inclusion protein became a soluble form with nature conformation. The purified protein retained it's biological activity as assayed by its ability to phosphatidylinositol 4,5-bisphosphate (PIP2). Conclusions: The PH domain was successfully expressed and purified, which can be used to further study for its structure and function.

关 键 词：丝苏氨酸蛋白激酶B PH结构域 原核表达 蛋白纯化 

分 类 号：Q55[生物学—生物化学]