Expression, purification and characterization of ROP6_6-178 GTPase from Arabidopsis thaliana

The unique type of GTPases in plants, termed ROPs, are the small GTP-binding proteins involved in signal transduction which play important roles in regulation of hormonal response pathway, cell polarity, defense from plant pathogens, etc. In order to explore the regulation mechanism of AtROPs involved in, the purified ROPs were needed to explore the interactions of ROP GTPases with their regulators and effectors. In this study, the first ROP GTPase from Arabidopsis thaliana, AtROP6_6-178 was successfully expressed in Escherichia coli and obtained in high quality and purity through affinity chromatography and gel-filtration chromatography. The resultant protein was identified as a single band of 19 kDa in SDS-PAGE and was confirmed to be active to interact with guanine nucleotides through the fluorescence-based assay. The intrinsic tryptophan fluorescence intensity of AtROP6_6-178 was enhanced upon interacting with either GDP or GTP. Meanwhile, the equilibrium dissociation constants of AtROP6_6-178 with fluorescent guanine nucleotide analogue mantGDP and mantGTP were determined to be 0.0721 μM and 0.0422 μM, respectively, based on fluorescence polarization.