title: Expression, purification and guanine nucleotide binding characterization of Arabidopsis RabE1d13-185 GTPase.
abstract: Arabidopsis RabE1d subclass plays important plant-specific functions in plant growth and development, response to ethylene and defence to plant pathogen, besides their basic cellular role in membrane trafficking. In this study, we present the expression, purification, and characterization of the recombinant core domain of AtRabE1d13-185. AtRabE1d13-185 was successfully expressed in Escherichia coli and purified via two-step nickel affinity chromatography followed by gel filtration, and identified single band in SDS-PAGE. The resultant protein was functionally active, as determined by interaction with guanine nucleotide by a fluorescence-based assay. The intrinsic tryptophan of AtRabE1d13-185 showed fluorescence resonance energy transfer (FRET) effect upon forming complex with fluorescent methylanthraniloyl (mant)-GDP, but quenched when binding with non-labelled guanine nucleotide. The association rate of mantGDP with AtRabE1d13-185 was determined to be 3.48 × 10(7) s(-1) M(-1). The dissociation rates of GDP and mantGDP from the complex with AtRabE1d13-185 were similar. The koff values were determined to be 4.02 × 10(-4) s(-1) based on the FRET effect for the AtRabE1d13-185:GDP and 5.41 × 10(-4) s(-1) for mantGDP excited directly.
authors.affiliation: Key Laboratory of Plant Biotechnology of Shandong Province, College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Key Laboratory of Plant Biotechnology of Shandong Province, College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China.
authors.forename: Lingling
authors.initials: L
authors.lastname: Tan
authors.affiliation: Key Laboratory of Plant Biotechnology of Shandong Province, College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China.
authors.forename: Chun-Hai
authors.initials: CH
authors.lastname: Dong
authors.affiliation: Key Laboratory of Plant Biotechnology of Shandong Province, College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China. Electronic address: tsfhxm@hotmail.com.
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Hou
mesh_terms: D029681:Arabidopsis Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000737:chemistry; D004926:Escherichia coli; D015870:Gene Expression; D006153:Guanosine Diphosphate / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000737:chemistry*; D007700:Kinetics; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011485:Protein Binding; D020691:rab GTP-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D029681:Arabidopsis Proteins; D010446:Peptide Fragments; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D010450:Endopeptidases; C099488:TEV protease; C000611663:RabE1d protein (13-185), Arabidopsis; D020691:rab GTP-Binding Proteins
keywords: AtRabE1d(13)(-185); Fluorescence-based assay; Protein purification