小G蛋白RhoA活性检测方法的优化

摘要

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 in-fluence the following measurement of active RhoA efficiently .We totally synthesized the sequence of RBD through optimization of mam-malian codons to prokaryotic codons , and then cloned RBD into pGEX 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 opti-mized pulldown experiment system would provide practical technique for the role of RhoA in cell migration .%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。