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.

• PMID: 16313173
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Shi C, Shin YO, Hanson J, Cass B, Loewen MC, Durocher Y

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