Recombinant Expression and Functional Characterization of Mouse Olfactory Receptor mOR256-17 in Mammalian Cells

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• 作者：Thamani Dahoun ; Luigino Grasso ; Horst Vogel ; Horst Pick
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：August 23, 2011
• 年：2011
• 卷：50
• 期：33
• 页码：7228-7235
• 全文大小：380K
• 年卷期：v.50,no.33(August 23, 2011)
• ISSN：1520-4995

文摘

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 10⁶ 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.