蛋白激酶BIN2的纯化及活性分析  

Purification and Activity Analysis of Tag-free Protein Kinase BIN2

作　　者：袁敏 齐玉荣[2] 王瑞菊[2] YUAN Min QI Yu-rong WANG Rui-ju(Center for Genomics and Computational Biology, College of Life Sciences, North China University of Science and Technology, Tangshan 063000 College of Life Sciences, Hebei Normal University, Shijiazhuang 050024)

机构地区：[1]华北理工大学生命科学学院基因组学与计算生物学研究中心,唐山063000 [2]河北师范大学生命科学学院,石家庄050024

出　　处：《生物技术通报》2017年第7期145-149,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31401212;31201063);河北省自然科学基金(C2014209134;C2012205042)

摘　　要：BIN2是一种丝氨酸/苏氨酸蛋白激酶,在油菜素内酯(BR)信号转导中发挥重要作用。为了获得具有激酶活性且不带标签的BIN2蛋白,将BIN2基因构建到带eXact标签的pPAL8表达载体上,获得BIN2基因的原核表达载体eXact-BIN2,转化大肠杆菌表达菌株BL21(DE3)。摸索合适的诱导条件后成功诱导出51 kD的eXact-BIN2蛋白。经eXact纯化介质纯化并切除标签,获得质量较高、不带标签的BIN2蛋白。该BIN2蛋白具有较高的激酶活性,能够在体外磷酸化MBP-BZR1蛋白。BIN2 is a kind of Ser/Thr protein kinase,and plays important roles in BR signal transduction pathway. In order to obtain theactive and tag-free BIN2 protein,the BIN2 gene was cloned into the prokaryotic expression vector pPAL8 to obtain BIN2-pPAL8 expressionvector. The correct plasmid BIN2-pPAL8 was transformed into E.coli expression strain BL21（DE3）. After exploring the proper inductionconditions,the 51 kD eXact-BIN2 protein was successfully induced and purified using e Xact purification system. The high-quality and tag-free BIN2 protein was obtained through cleaving the e Xact tag. The purified BIN2 protein is highly active,which could phosphorylate MBP-BZR1 in vitro.

关 键 词：BIN2 磷酸化 蛋白激酶 蛋白纯化 

分 类 号：Q55[生物学—生物化学]