蛋白激酶BIN2的纯化及活性分析

Purification and Activity Analysis of Tag-free Protein Kinase BIN2

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.