在大肠杆菌中高效表达可用于检测人源Rap1活性RapBD蛋白方法的建立

Expression of RapBD in Escherichia coli for detecting human Rap1 activity

【背景】Rap1是一种小GTP酶,其活性的检测方法少,目前主要依赖试剂盒,检测成本太高。而Rap1下游效应蛋白RalGDS具有Rap1结合结构域(Rap binding domain,RapBD),该结构域能与有活性的GTP-Rap1特异性结合。【目的】利用大肠杆菌外源表达GST-RapBD融合蛋白,建立经济的检测人源Rap1活性的方法。【方法】合成RapBD基因序列,插入pGEX-4T-1载体,使该质粒表达GST-RapBD融合蛋白,再利用GST亲和树脂结合大肠杆菌中表达的GST-RapBD融合蛋白,最后利用GST-RapBD融合蛋白Pulldown检测GTP-Rap1。【结果】建立了检测人源Rap1活性的方法。【结论】序列优化使得pGEX-4T-1载体在大肠杆菌中高效表达能特异性结合人源GTP-Rap1且带有GST标签的RapBD蛋白,提高了Pulldown实验检测GTP-Rap1的效率,降低了检测人源小G蛋白Rap1活性的成本。

[Background] Rap1 is a kind of small GTPase, and the method of its activity detection is very scanty. At present, the method mainly depends on commercialized kit, resulting in the high cost. RalGDS has the Rap binding domain（RapBD）, which can bind to GTP-Rap1 specifically. [Objective] Establish an inexpensive method of detecting human Rap1 activity by exogenous GST-RapBD fusion protein expressed in Escherichia coli. [Methods] We constructed the plasmid with pGEX-4 T-1 vector expressing GST-RapBD fusion protein in E. coli, and then combined GST-RapBD with GST affinity resin. Finally, we used GST Pulldown assay to detect Rap1 activity. [Results] The method of detecting human Rap1 activity was established successfully. [Conclusion] Sequence optimization made pGEX-4 T-1 highly express the GST tagged RapBD protein in E. coli, which increased the efficiency and decreased the cost of Pulldown assay to detect GTP-Rap1.