Abstract: 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 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 optimized pulldown experiment system would provide practical technique for the role of RhoA in cell migration. 

Key words: RhoA, codon optimization, GST-RBD pulldown