title: Expression, purification and characterization of ROP66-178GTPase from Arabidopsis thaliana.
abstract: 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, AtROP66-178was 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 AtROP66-178was enhanced upon interacting with either GDP or GTP. Meanwhile, the equilibrium dissociation constants of AtROP66-178with fluorescent guanine nucleotide analogue mantGDP and mantGTP were determined to be 0.0721 μM and 0.0422 μM, respectively, based on fluorescence polarization.
authors: Key Laboratory of Plant Biotechnology of Shandong Province, College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China.
authors: Yongheng
authors: Y
authors: Rong
authors: Key Laboratory of Plant Biotechnology of Shandong Province, College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China.
authors: Kun
authors: K
authors: Wang
authors: Key Laboratory of Plant Biotechnology of Shandong Province, College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China.
authors: Renxing
authors: R
authors: Shi
authors: Key Laboratory of Plant Biotechnology of Shandong Province, College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China. Electronic address: tsfhxm@hotmail.com.
authors: Xiaomin
authors: X
authors: Hou
authors: Key Laboratory of Plant Biotechnology of Shandong Province, College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China.
authors: Chun-Hai
authors: CH
authors: Dong
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D006153:Guanosine Diphosphate / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000737:chemistry*; D020559:Monomeric GTP-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D029681:Arabidopsis Proteins; C518506:ROP6 protein, Arabidopsis; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020559:Monomeric GTP-Binding Proteins
keywords: AtROP6; Fluorescence-based assay; Protein purification