Purification and characterization of a recombinant G-protein-coupled receptor, Saccharomyces cerevisiae Ste2p, transiently expressed in HEK293 EBNA1 cells.
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.
Shi
Chunhua
C
Plant Biotechnology Institute, National Research Council of Canada, and Department of Biochemistry, University of Saskatchewan, 110 Gymnasium Place, Saskatoon, Saskatchewan S7N 0W9, Canada.
Shin
Youn-Ok
YO
Hanson
John
J
Cass
Brian
B
Loewen
Michele C
MC
Durocher
Yves
Y
D002478:Cells, Cultured; D006801:Humans; D000072235:Mating Factor; D010455:Peptides / Q000378:metabolism; D010675:Pheromones / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D051964:Receptors, Mating Factor / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D014162:Transfection
D010455:Peptides; D010675:Pheromones; D043562:Receptors, G-Protein-Coupled; D051964:Receptors, Mating Factor; D011993:Recombinant Fusion Proteins; C498031:STE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000072235:Mating Factor