泛素连接酶LNX1的表达、活性测定和功能初探  

Expression,Activity Determination of Ubiquitin Ligase LNX1 and Primary Study on its Function

作　　者：郭正光[1] 高友鹤[1] 

机构地区：[1]中国医学科学院基础医学研究所生理学与病理生理学系医学分子生物学国家重点实验室,北京100005

出　　处：《生物技术通讯》2012年第4期523-526,共4页Letters in Biotechnology

基　　金：国家自然科学基金杰出青年基金(30725009)

摘　　要：目的:在大肠杆菌中表达重组人泛素连接酶LNX1,并研究其对钾离子通道蛋白Kv1.4的泛素化作用。方法:构建人LNX1重组蛋白原核表达载体pGEX-LNX1,在大肠杆菌中通过IPTG诱导表达重组蛋白,表达产物经GSTrap FF纯化,并通过体外泛素化(in vitroubiquitination)方法测定其泛素连接酶活性,同样用体外泛素化方法研究其对含有Kv1.4的C端的人工底物的泛素化。结果:获得了纯化的有泛素连接酶活性的重组人LNX1蛋白,重组LNX1可以在体外泛素化体系中泛素化含有Kv1.4的C端的人工底物。结论:重组人LNX1原核表达成功,具有泛素连接酶活性,并催化Kv1.4的泛素化。Objective： To express human recombinant ubiquitin ligase LNXl（ligand of Numb protein X 1） in E. eoli, and to study its role in the ubiquinatination of potassium channel protein Kvl.4. Methods： The expression plasmid pGEX-LNX1 was constructed, the recombinant LNX1 were expressed in E.coli by induction with IPTG and purified by GSTrap FF. The in vitro ubiquitination method was used to evaluating its ubiquitin ligase activity, and also ws used to identify the ubiquitnation of artifitial substrate containing the C-terminal of Kvl.4 by LNX1. Results： The purified and ubiqutin ligase-actived recombinant human LNX1 was successfully acquired. Artificial substrate containing the C terminal of Kvl.4 could be ubiquitinated by LNX1 in in vitro ubiquitination system. Conclusion： Human recombinant LNX1 was expressed and showed good ubiquitin ligase activity, which can mediate the ubiquinatination of Kvl.4.

关 键 词：泛素连接酶 表达 酶活性测定 LNX1 Kv1.4 

分 类 号：Q78[生物学—分子生物学] Q55