小G蛋白RhoA活性检测方法的优化

Optimization of the method for measurement of the activity of small GTP binding protein RhoA

Rho蛋白家族的小G蛋白是调节细胞迁移的关键蛋白,具有GTP酶活性。RhoA是该蛋白家族的重要成员,通过其催化活性来调节肌动蛋白的聚集和收缩,在肿瘤迁移和侵袭中发挥重要作用。所以,RhoA的GTP酶活性检测对于研究细胞迁移的机制,以及靶向RhoA的治疗策略具有重要意义。RhoA下游效应子Rhotekin基因(RhoA effector)具有RhoA结合结构域(RhoA binding domain,RBD),该RBD蛋白结构域能与活化状态的GTP-RhoA特异结合。通常用外源表达的RBD蛋白pulldown结合GTP-RhoA的蛋白量,作为RhoA活性检测方法。但是,哺乳动物来源的RBD蛋白在原核细胞中的表达量很低,使得pulldown结合GTP-RhoA蛋白量的效率低下,从而不利于RhoA的活性检测。因此,目的在于提高RBD蛋白在大肠杆菌中的产量,提高RBD蛋白pulldown结合GTP-RhoA的效率,从而优化RhoA活性检测方法。通过密码子优化技术对RBD基因进行改良,提高其蛋白在大肠杆菌中的表达量,优化RBD蛋白pulldown实验检测GTP-RhoA的效率。优化后的RBD与GST标签蛋白融合后在大肠杆菌中高效表达,并且能够特异性地与GTP-RhoA结合。成功地构建了具有较高表达水平的GST-RBD载体,并优化了传统的GST-RBD pulldown检测体系,为进一步深入研究RhoA在细胞迁移中的功能提供了一种有效的技术手段。

The small G protein of Rho GTPase family was key regulator in cell migration. As the important member of Rho GTPase family,RhoA regulates the aggregation and contraction of actin,which plays a vital role for cell migration. Monitoring the active RhoA is essential to study RhoA＇s function in vivo and as a potential target for tumor therapy. RhoA binding domain（ RBD） of Rhotekin,a RhoA effector protein interacts only with active RhoA. RBD pulldown assay is a common assay and usually used to detect GTP-RhoA in vivo. However,gene sequence of RBD with original codon ectopically expressed in E. coli has a relative low expression level which influence the following measurement of active RhoA efficiently. We totally synthesized the sequence of RBD through optimization of mammalian codons to prokaryotic codons,and then cloned RBD into p GEX prokaryotic expression vector. The GST tagged RBD protein was highly expressed in E. coli,and specifically bound to the active form of RhoA（ GTP-RhoA）. The GST-RBD construct and the optimized pulldown experiment system would provide practical technique for the role of RhoA in cell migration.