泛素连接酶LNX1的表达、活性测定和功能初探

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。

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

Objective: To express human recombinant ubiquitin ligase LNX1(ligand of Numb protein X 1) in E.coli,and to study its role in the ubiquinatination of potassium channel protein Kv1.4.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 Kv1.4 by LNX1.Results: The purified and ubiqutin ligase-actived recombinant human LNX1 was successfully acquired.Artificial substrate containing the C terminal of Kv1.4 could be ubiquitinated by LNX1 in in vitro ubiquitination system.Conclusion: Human recombinant LNX1 was expressed and showed good ubiquitin ligase activity,which can medi ate the ubiquinatination of Kv1.4.