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Purification and Characterization of a Recombinant G-Protein-Coupled Receptor, Saccharomyces cerevisiae Ste2p, Transiently Expressed in HEK293 EBNA1 Cells

Shi, Chunhua ; Shin, Youn-Ok ; Hanson, John ; Cass, Brian ; Loewen, Michele C ; Durocher, Yves

Biochemistry (Easton), 2005-12, Vol.44 (48), p.15705-15714 [Periódico revisado por pares]

United States: American Chemical Society

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Título:
Purification and Characterization of a Recombinant G-Protein-Coupled Receptor, Saccharomyces cerevisiae Ste2p, Transiently Expressed in HEK293 EBNA1 Cells
Autor: Shi, Chunhua ; Shin, Youn-Ok ; Hanson, John ; Cass, Brian ; Loewen, Michele C ; Durocher, Yves
Assuntos: Cells, Cultured ; Humans ; Mating Factor ; Peptides - metabolism ; Pheromones - metabolism ; Receptors, G-Protein-Coupled - biosynthesis ; Receptors, G-Protein-Coupled - genetics ; Receptors, G-Protein-Coupled - isolation & purification ; Receptors, Mating Factor - biosynthesis ; Receptors, Mating Factor - genetics ; Receptors, Mating Factor - isolation & purification ; Recombinant Fusion Proteins - biosynthesis ; Recombinant Fusion Proteins - isolation & purification ; Saccharomyces cerevisiae ; Saccharomyces cerevisiae Proteins - biosynthesis ; Saccharomyces cerevisiae Proteins - genetics ; Saccharomyces cerevisiae Proteins - isolation & purification ; Transfection
É parte de: Biochemistry (Easton), 2005-12, Vol.44 (48), p.15705-15714
Notas: ark:/67375/TPS-XDJS7BH8-C
istex:A2243E282723FB8C3E7442149F26244690D12EF3
This work was supported by NRC Canada, Genomics Health Initiative Grant 49-GH2P6 to M.C.L. and Y.D., and by a Saskatchewan Synchrotron Institute Fellowship to C.S. and Y.-O.S.
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Descrição: 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 (∼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 α-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. K D 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 α-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.
Editor: United States: American Chemical Society
Idioma: Inglês

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Purification and Characterization of a Recombinant G-Protein-Coupled Receptor, Saccharomyces cerevisiae Ste2p, Transiently Expressed in HEK293 EBNA1 Cells

Shi, Chunhua ; Shin, Youn-Ok ; Hanson, John ; Cass, Brian ; Loewen, Michele C ; Durocher, Yves

United States: American Chemical Society

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