拟南芥蛋白激酶基因BIN2的原核表达纯化及其酶活性分析

Expression,Purification and Characterization of Arabidopsis BIN2

为了研究蛋白激酶BIN2(BR-insensitive-2)的底物及其在BR信号通路中的作用机制,通过One Step Cloning方法将BIN2基因克隆至SUMO表达载体,利用SUMO标签促进目的蛋白表达和折叠的特性,对BIN2蛋白进行原核表达和纯化,进而检测BIN2的激酶活性。结果表明,纯化得到的BIN2具有自身磷酸化活性,并测得其酶活大小和动力学曲线。为进一步研究BIN2蛋白的作用机制提供了基础材料。

In order to study the target substrates of protein kinase BIN2 and its roles in the BR signaling pathway,we cloned BIN2 gene into the SUMO expression vector by one step cloning method. BIN2 was expressed and purified as a SUMO fusion protein, which facilitated folding and stabilization of heterolo- gously expressed BIN2. Further activity analysis showed that purified BIN2 was functional and got auto- phosphorylated in vitro. In this study, the enzyme activity and kinetics curve of BIN2 were acquired as well. This laid a foundation for further analysis of the function and targets of BIN2.