| Title: | "Purification and characterization of a recombinant G-protein-coupled receptor, Saccharomyces cerevisiae Ste2p, transiently expressed in HEK293 EBNA1 cells" |
| Author(s): | Shi C; Shin YO; Hanson J; Cass B; Loewen MC; Durocher Y; |
| Address: | "Plant Biotechnology Institute, National Research Council of Canada, and Department of Biochemistry, University of Saskatchewan, 110 Gymnasium Place, Saskatoon, Saskatchewan S7N 0W9, Canada" |
| ISSN/ISBN: | 0006-2960 (Print) 0006-2960 (Linking) |
| Abstract: | "The production of milligram quantities of purified, active, folded membrane protein from heterologous expression systems remains a general challenge due to intrinsically low expression levels, misfolding, and instability. Here we report the overexpression and purification of milligram quantities of functional Saccharomyces cerevisiae G-protein-coupled receptor, Ste2p, from transiently transfected human embryonic kidney 293 EBNA1 cells. Fluorescent microscopy indicates localization of Ste2p-GFP and Fc-Ste2p-GFP fusion receptors to the cell membrane. Up to 2 mg (approximately 10 pmol/million cells) of the Fc-Ste2p-GFP fusion and 1 mg of a Ste2p-Strep-TagII/(His)8-tagged version were purified per liter of culture following protein A-Sepharose and Talon metal affinity chromatography, respectively. Two distinct fluorescent labels, the hydrophobic 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) and the more hydrophilic fluorescein-5-maleimide (FM), were individually attached to the C-terminus of the alpha-mating factor ligand by addition of a reactive cysteine residue to produce active fluorescent pheromones. In vitro fluorescent ligand binding assays demonstrated that a high percentage of the recombinant purified receptor is correctly folded and able to bind ligand. KD values of 34 +/- 3 and 300 +/- 20 nM were observed respectively for the CPM- and FM-labeled ligands. These results combined with blue-shifted emission peaks and loss of fluorescent quenching observed for both fluorescent-labeled Cys alpha-factors when bound to receptor support a model in which the C-terminus of the ligand is packed in a hydrophobic pocket at the interface between the transmembrane and extracellular loop domains. Overall, we present an efficient system for recombinant production of milligram quantities of purified Ste2p in a biologically active form with applications to future structure and functional studies" |
| Keywords: | "Cells, Cultured Humans Mating Factor Peptides/metabolism Pheromones/metabolism Receptors, G-Protein-Coupled/biosynthesis/genetics/*isolation & purification Receptors, Mating Factor/biosynthesis/genetics/*isolation & purification Recombinant Fusion Protein;" |
| Notes: | "MedlineShi, Chunhua Shin, Youn-Ok Hanson, John Cass, Brian Loewen, Michele C Durocher, Yves eng Research Support, Non-U.S. Gov't 2005/11/30 Biochemistry. 2005 Dec 6; 44(48):15705-14. doi: 10.1021/bi051292p" |
