Overexpression, Solubization and Purification of Rat and Human Olfactory Receptors

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Abstract
The superfamily of olfactory receptor genes, whose products are thought to be activated by odorant gands, is critical for odor recognition. Two olfactory receptors, olp4 from rat and OR17–4 from human, were overexpressed in Sf9 insect cells. The presence of the proteins in cell membranes was monitored by immunoblotting with peptide-specific polyclonal antibodies directed against the C-terminal sequences of these receptors and with a mAb against an N-terminal octapeptide epitope tag. A DNA sequence that codes for a His6 tag, which binds tightly to a Ni2+-chelate-affinity column, was incorporated into the N-termini of both genes. The expressed olfactory receptors were found mainly in the cell-membrane fraction. The proteins were difficult to solubize by many detergents and only lysophosphatidylchone was found to be both suitable for efficient solubization of the overexpressed olfactory receptors and compatible with the purification system used. After solubization, the olfactory receptors were purified to near homogeneity by affinity chromatography on nickel nitrilotriacetic acid resin and by cation-exchange chroma-tography. Electrophoresis of the purified proteins and visuazation with Coomassie Blue staining or by immunoblotting with specific antibodies, revealed bands of 32, 69 and 94 kDa, which were identified as the monomeric, dimeric and trimeric forms of the receptor proteins. The ogomeric forms were resistant to reduction and alkylation, and are therefore thought to be held together by non-covalent hydrophobic interactions that are resistant to SDS. This finding is similar to previous observations for other guanine-nucleotide-binding-regulatory-protein-coupled receptors. Reconstitution in phosphopid vesicles showed that the purified olfactory receptors insert specifically into the pid bilayer. This provides a means to study functional reconstitution with putative transduction components such as olfactory guanine-nucleo-tide-binding-regulatory protein.