Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4

doi:10.1073/pnas.0811089106

Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4

^aDepartment of Biological Engineering, and Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139-4307;
^bBiacore Life Sciences, GE Healthcare, 800 Centennial Avenue, Piscataway, NJ 08854; and
^cInstitut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015, Lausanne, Switzerland

Abstract

Although understanding of the olfactory system has progressed at the level of downstream receptor signaling and the wiring of olfactory neurons, the system remains poorly understood at the molecular level of the receptors and their interaction with and recognition of odorant ligands. The structure and functional mechanisms of these receptors still remain a tantalizing enigma, because numerous previous attempts at the large-scale production of functional olfactory receptors (ORs) have not been successful to date. To investigate the elusive biochemistry and molecular mechanisms of olfaction, we have developed a mammalian expression system for the large-scale production and purification of a functional OR protein in milligram quantities. Here, we report the study of human OR17-4 (hOR17-4) purified from a HEK293S tetracycline-inducible system. Scale-up of production yield was achieved through suspension culture in a bioreactor, which enabled the preparation of >10 mg of monomeric hOR17-4 receptor after immunoaffinity and size exclusion chromatography, with expression yields reaching 3 mg/L of culture medium. Several key post-translational modifications were identified using MS, and CD spectroscopy showed the receptor to be ≈50% α-helix, similar to other recently determined G protein-coupled receptor structures. Detergent-solubilized hOR17-4 specifically bound its known activating odorants lilial and floralozone in vitro, as measured by surface plasmon resonance. The hOR17-4 also recognized specific odorants in heterologous cells as determined by calcium ion mobilization. Our system is feasible for the production of large quantities of OR necessary for structural and functional analyses and research into OR biosensor devices.

Footnotes

¹To whom correspondence may be addressed. E-mail: cookb{at}mit.edu or shuguang{at}mit.edu
Edited by Alexander Varshavsky, California Institute of Technology, Pasadena, CA, and approved May 4, 2009
Author contributions: B.L.C., D.S., L.K., J.G.-B., K.H., H.P., H.V., and S.Z. designed research; B.L.C., D.S., L.K., J.G.-B., M.V., A.P.B., K.H., and H.P. performed research; K.H., H.P., and H.V. contributed new reagents/analytic tools; B.L.C., D.S., L.K., J.G.-B., M.V., K.H., H.P., H.V., and S.Z. analyzed data; and B.L.C., L.K., J.G.-B., H.P., and S.Z. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0811089106/DCSupplemental.