Expression of an Olfactory Receptor in Escherichia coli: Purification, Reconstitution, and Ligand Binding ² Hans Kiefer,* ,‡,§ Ju ¨rgen Krieger, | John D. Olszewski, ⊥,∇ Gunnar von Heijne, ‡ Glenn D. Prestwich, ⊥,# and Heinz Breer | Stockholm UniVersity, Department of Biochemistry, S-106 91 Stockholm, Sweden, UniVersity of StuttgartsHohenheim, Institute of Zoophysiology, D-70 593 Stuttgart, Germany, and State UniVersity of New York, Department of Biochemistry, Stony Brook, New York 11794-3400 ReceiVed May 20, 1996; ReVised Manuscript ReceiVed September 30, 1996 X ABSTRACT: An olfactory receptor has been expressed in bacterial cells as a fusion protein with glutathione S-transferase (GST). Overexpression of receptor protein yielding about 10% of the cell protein was achieved with mutants lacking the N-terminus and the first transmembrane region or with mutants carrying three positively charged residues in the first intracellular loop. The overexpressed fusion protein accumulated in inclusion bodies and could be solubilized in detergent. It was purified by metal chelation chromatography based on a C-terminal 6-histidine tag, and the GST portion was removed after proteolytic cleavage. The purified receptor was reconstituted into lipid vesicles and specific binding of odor ligands was shown by photoaffinity labeling and tryptophan fluorescence measurements. Thus, for the first time, an odorant receptor/ligand pair becomes available in large amounts for biophysical and screening studies. The olfactory system of vertebrates recognizes and dis- criminates thousands of odorous compounds. The enormous capacity of chemical recognition is probably based on a large family of olfactory receptors comprised of several hundreds or a thousand subtypes (Buck & Axel, 1991). These receptors are predicted to contain seven transmembrane segments, consistent with the view that odorant signals are transduced via G-protein-coupled cascades in olfactory sensory neurons (Reed et al., 1992; Breer et al., 1994). Different receptor types are presumed to be tuned to odorous compounds with distinct chemical determinants, and odor coding may be accomplished by expression of one or a few receptor types in individual chemosensory neurons (Lancet et al., 1987; Shepherd, 1994; Mori & Yoshihara, 1995). Soluble ligand-specific odorant binding proteins have also been implicated in the decoding of odor and in signal transduction (Pelosi, 1994; Du & Prestwich, 1995; Prestwich et al., 1995). Most of the current information on olfactory receptors is based on genes and mRNA studies; the knowledge about the nature of the actual receptor proteins is still very limited. Using the baculovirus/Sf9-system several receptor types have been heterologously expressed (Raming et al., 1993; Gat et al., 1994; Nekrasova et al., 1996). Attempts have been made to determine the ligand specificity of individual receptor types (Raming et al., 1993; Meinken, 1995); however, the functional assays used would be impractical to implement for screening large arrays of odor ligands and receptor types. Several G-protein-coupled receptors (GPCRs) 1 have been expressed in bacterial cells, and when inserted in the inner membrane displayed all the ligand binding properties ob- served in mammalian cells. However, high-level expression of membrane proteins, and especially of GPCRs, in bacteria appear to be difficult (Schertler, 1992; Grisshammer & Tate, 1995), probably due to the fact that the cells cannot tolerate large amounts of the foreign protein within their membranes. Milligram quantities of purified membrane protein which are required for biophysical studies or attempts to crystallize the protein have only been obtained by overexpressing the protein in E. coli as inclusion bodies (IBs), i.e., not integrated in the cell membrane. Proteins derived from IBs are non- native and have to be folded to the native state. This has been achieved for many soluble, but only for a few membrane proteins (Matsuyama et al., 1992; Fiermonte et al., 1993; Efimov et al., 1994); for an overview, see Grisshammer and Tate (1995). As far as we are aware, no GPCR has previously been expressed at high level in bacteria. Purification of bacterially expressed GPCRs has only been reported in one case (Tucker & Grisshammer, 1996). In this recent study, microgram amounts of the neurotensin receptor were purified from E. coli. Because of the low expression level, the protein was affinity-tagged, and purification was achieved in two highly efficient chromatographic steps. ² This work was supported by the Deutsche Forschungsgemeinschaft, grant Br 712/16-1, the Human Science Frontier Program, the Fond der Chemischen Industrie, the Bundesministerium fu ¨r Forschung und Technologie (Grant 0319325A), and by a grant of the NIH (NS 29632) to G.D.P. * Author to whom correspondence should be addressed. Tel: +49 711 459 2221. FAX: +49 711 459 2238. ‡ Stockholm University. § Present address: University of Hohenheim, Department of Micro- biology, Garbenstrasse 30, D-70 599 Stuttgart, Germany. | University of StuttgartsHohenheim. ⊥ State University of New York. ∇ Present address: Wyeth-Ayerst Laboratories, 401 North Middle- town Road, Pearl River, NY 10965. # Present address: Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112. X Abstract published in AdVance ACS Abstracts, November 15, 1996. 1 Abbreviations: BDCA, 4-benzoyldihydrocinnamaldehyde; DTT, dithiothreitol; GPCR, G protein-coupled receptor; GST, glutathione S-transferase; IB, inclusion body; OR, olfactory receptor; PC, phos- phatidylcholine; PCC, pyridinium chlorochromate; PMSF, phenyl- methylsulfonyl fluoride; sarcosyl, N-lauroylsarcosine; TBS, Tris- buffered saline; TM, transmembrane. 16077 Biochemistry 1996, 35, 16077-16084 S0006-2960(96)01206-8 CCC: $12.00 © 1996 American Chemical Society