Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4
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 a...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | en_US |
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United States National Academy of Sciences
2010
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| Online Access: | http://hdl.handle.net/1721.1/52554 |
| _version_ | 1811097740378112000 |
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| author | Berke, Allison P. Vanberghem, Melanie Graveland-Bikker, Johanna Kaiser, Liselotte Steuerwald, Dirk Cook, Brian L. Zhang, Shuguang Vogel, Horst Pick, Horst Herlihy, Kara |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Berke, Allison P. Vanberghem, Melanie Graveland-Bikker, Johanna Kaiser, Liselotte Steuerwald, Dirk Cook, Brian L. Zhang, Shuguang Vogel, Horst Pick, Horst Herlihy, Kara |
| author_sort | Berke, Allison P. |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T17:04:11Z |
| format | Article |
| id | mit-1721.1/52554 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T17:04:11Z |
| publishDate | 2010 |
| publisher | United States National Academy of Sciences |
| record_format | dspace |
| spelling | mit-1721.1/525542022-09-29T23:27:13Z Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4 Berke, Allison P. Vanberghem, Melanie Graveland-Bikker, Johanna Kaiser, Liselotte Steuerwald, Dirk Cook, Brian L. Zhang, Shuguang Vogel, Horst Pick, Horst Herlihy, Kara Massachusetts Institute of Technology. Department of Biological Engineering Zhang, Shuguang Berke, Allison P. Vanberghem, Melanie Graveland-Bikker, Johanna Kaiser, Liselotte Steuerwald, Dirk Cook, Brian L. Zhang, Shuguang 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. ROHM Corporation, Kyoto, Japan 2010-03-12T20:51:38Z 2010-03-12T20:51:38Z 2009-05 2008-11 Article http://purl.org/eprint/type/JournalArticle 1091-6490 0027-8424 http://hdl.handle.net/1721.1/52554 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 en_US http://dx.doi.org/10.1073/pnas.0811089106 Proceedings of the National Academy of Sciences of the United States of America Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf United States National Academy of Sciences PNAS |
| spellingShingle | Berke, Allison P. Vanberghem, Melanie Graveland-Bikker, Johanna Kaiser, Liselotte Steuerwald, Dirk Cook, Brian L. Zhang, Shuguang Vogel, Horst Pick, Horst Herlihy, Kara Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4 |
| title | Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4 |
| title_full | Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4 |
| title_fullStr | Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4 |
| title_full_unstemmed | Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4 |
| title_short | Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4 |
| title_sort | large scale production and study of a synthetic g protein coupled receptor human olfactory receptor 17 4 |
| url | http://hdl.handle.net/1721.1/52554 |
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