Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4
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Cook-2009-Large-scale producti.pdf
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1.49 MB
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Author(s)
Berke, Allison P.
Vanberghem, Melanie
Graveland-Bikker, Johanna
Kaiser, Liselotte
Steuerwald, Dirk
Cook, Brian L.
Zhang, Shuguang
Vogel, Horst
Pick, Horst
Herlihy, Kara
Date Issued
May 2009
Journal
Proceedings of the National Academy of Sciences of the United States of America
Publisher
United States National Academy of Sciences
Citation
Cook, Brian L et al. “Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4.” Proceedings of the National Academy of Sciences 106.29 (2009): 11925-11930. © 2009 National Academy of Sciences
Version
Final published version
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.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
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DOI of Published Version
http://dx.doi.org/10.1073/pnas.0811089106
