Study of a Synthetic Human Olfactory Receptor 17-4: Expression and Purification from an Inducible Mammalian Cell Line
In order to begin to study the structural and functional mechanisms of olfactory receptors, methods for milligram-scale purification are required. Here we demonstrate the production and expression of a synthetically engineered human olfactory receptor hOR17-4 gene in a stable tetracycline-inducible...
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| Format: | Article |
| Language: | en_US |
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Public Library of Science
2010
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| Online Access: | http://hdl.handle.net/1721.1/55353 |
| _version_ | 1826198330350239744 |
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| author | Zhang, Shuguang Chung, Hyeyoun Ernberg, Karin E. Cook, Brian L. |
| author2 | Massachusetts Institute of Technology. Center for Biomedical Engineering |
| author_facet | Massachusetts Institute of Technology. Center for Biomedical Engineering Zhang, Shuguang Chung, Hyeyoun Ernberg, Karin E. Cook, Brian L. |
| author_sort | Zhang, Shuguang |
| collection | MIT |
| description | In order to begin to study the structural and functional mechanisms of olfactory receptors, methods for milligram-scale purification are required. Here we demonstrate the production and expression of a synthetically engineered human olfactory receptor hOR17-4 gene in a stable tetracycline-inducible mammalian cell line (HEK293S). The olfactory receptor gene was fabricated from scratch using PCR-based gene-assembly, which facilitated codon optimization and attachment of a 9-residue bovine rhodopsin affinity tag for detection and purification. Induction of adherent cultures with tetracycline together with sodium butyrate led to hOR17-4 expression levels of ~30 µg per 150 mm tissue culture plate. Fos-choline-based detergents proved highly capable of extracting the receptors, and fos-choline-14 (N-tetradecylphosphocholine) was selected for optimal solubilization and subsequent purification. Analysis by SDS-PAGE revealed both monomeric and dimeric receptor forms, as well as higher MW oligomeric species. A two-step purification method of immunoaffinity and size exclusion chromatography was optimized which enabled 0.13 milligrams of hOR17-4 monomer to be obtained at >90% purity. This high purity of hOR17-4 is not only suitable for secondary structural and functional analyses but also for subsequent crystallization trials. Thus, this system demonstrates the feasibility of purifying milligram quantities of the GPCR membrane protein hOR17-4 for fabrication of olfactory receptor-based bionic sensing device. |
| first_indexed | 2024-09-23T11:03:10Z |
| format | Article |
| id | mit-1721.1/55353 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T11:03:10Z |
| publishDate | 2010 |
| publisher | Public Library of Science |
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| spelling | mit-1721.1/553532022-09-27T16:45:50Z Study of a Synthetic Human Olfactory Receptor 17-4: Expression and Purification from an Inducible Mammalian Cell Line Zhang, Shuguang Chung, Hyeyoun Ernberg, Karin E. Cook, Brian L. Massachusetts Institute of Technology. Center for Biomedical Engineering Massachusetts Institute of Technology. Department of Biological Engineering Zhang, Shuguang Zhang, Shuguang Chung, Hyeyoun Ernberg, Karin E. Cook, Brian L. In order to begin to study the structural and functional mechanisms of olfactory receptors, methods for milligram-scale purification are required. Here we demonstrate the production and expression of a synthetically engineered human olfactory receptor hOR17-4 gene in a stable tetracycline-inducible mammalian cell line (HEK293S). The olfactory receptor gene was fabricated from scratch using PCR-based gene-assembly, which facilitated codon optimization and attachment of a 9-residue bovine rhodopsin affinity tag for detection and purification. Induction of adherent cultures with tetracycline together with sodium butyrate led to hOR17-4 expression levels of ~30 µg per 150 mm tissue culture plate. Fos-choline-based detergents proved highly capable of extracting the receptors, and fos-choline-14 (N-tetradecylphosphocholine) was selected for optimal solubilization and subsequent purification. Analysis by SDS-PAGE revealed both monomeric and dimeric receptor forms, as well as higher MW oligomeric species. A two-step purification method of immunoaffinity and size exclusion chromatography was optimized which enabled 0.13 milligrams of hOR17-4 monomer to be obtained at >90% purity. This high purity of hOR17-4 is not only suitable for secondary structural and functional analyses but also for subsequent crystallization trials. Thus, this system demonstrates the feasibility of purifying milligram quantities of the GPCR membrane protein hOR17-4 for fabrication of olfactory receptor-based bionic sensing device. ROHM Corporation, Kyoto, Japan 2010-05-28T19:33:55Z 2010-05-28T19:33:55Z 2008-08 2008-07 Article http://purl.org/eprint/type/JournalArticle 1932-6203 http://hdl.handle.net/1721.1/55353 Cook BL, Ernberg KE, Chung H, Zhang S (2008) Study of a Synthetic Human Olfactory Receptor 17-4: Expression and Purification from an Inducible Mammalian Cell Line. PLoS ONE 3(8): e2920. doi:10.1371/journal.pone.0002920 en_US http://dx.doi.org/10.1371/journal.pone.0002920 PLoS ONE 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 Public Library of Science PLoS |
| spellingShingle | Zhang, Shuguang Chung, Hyeyoun Ernberg, Karin E. Cook, Brian L. Study of a Synthetic Human Olfactory Receptor 17-4: Expression and Purification from an Inducible Mammalian Cell Line |
| title | Study of a Synthetic Human Olfactory Receptor 17-4: Expression and Purification from an Inducible Mammalian Cell Line |
| title_full | Study of a Synthetic Human Olfactory Receptor 17-4: Expression and Purification from an Inducible Mammalian Cell Line |
| title_fullStr | Study of a Synthetic Human Olfactory Receptor 17-4: Expression and Purification from an Inducible Mammalian Cell Line |
| title_full_unstemmed | Study of a Synthetic Human Olfactory Receptor 17-4: Expression and Purification from an Inducible Mammalian Cell Line |
| title_short | Study of a Synthetic Human Olfactory Receptor 17-4: Expression and Purification from an Inducible Mammalian Cell Line |
| title_sort | study of a synthetic human olfactory receptor 17 4 expression and purification from an inducible mammalian cell line |
| url | http://hdl.handle.net/1721.1/55353 |
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