title: Recombinant expression and functional characterization of mouse olfactory receptor mOR256-17 in mammalian cells.
abstract: Olfactory receptors (ORs) constitute the largest family of sensory membrane proteins in mammals. They play a key role within the olfactory system in recognizing and discriminating a nearly unlimited number of structurally diverse odorous molecules. The molecular basis of OR-mediated signal detection and transduction is poorly understood. This is due to difficulties in functional expression of ORs in high yields, preventing structural and biophysical studies at the level of the receptor protein. Here we report on recombinant expression of mouse receptor mOR256-17 yielding 10(6) ORs per cell in transiently transfected mammalian cells. For quantification and optimization of OR expression, we employed different fluorescent probes. Green fluorescent protein fused to the C-terminus of mOR256-17 allowed quantification of total cellular OR biosynthesis, and post-translational fluorescence labeling of a 12-amino acid polypeptide sequence at the N-terminus permitted the selective visualization and quantification of ORs at the plasma membrane using cell flow cytometry. Our dual-color labeling approach is generally applicable to quantification of membrane proteins for mammalian cell-based expression. By screening a large odorant compound library, we discovered a selective spectrum of potent mOR256-17-specific agonists essential for probing the receptor function for future scaled-up productions.
authors.affiliation: Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.
authors.forename: Thamani
authors.initials: T
authors.lastname: Dahoun
authors.forename: Luigino
authors.initials: L
authors.lastname: Grasso
authors.forename: Horst
authors.initials: H
authors.lastname: Vogel
authors.forename: Horst
authors.initials: H
authors.lastname: Pick
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018035:Receptors, Odorant / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C498563:Olfr256 protein, mouse; D018035:Receptors, Odorant; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins