High-Level Expression, Single-Step Immunoaffinity Purification and Characterization of Human Tetraspanin Membrane Protein CD81
The study of membrane protein structure and function requires their high-level expression and purification in fully functional form. We previously used a tetracycline-inducible stable mammalian cell line, HEK293S-TetR, for regulated high-level expression of G-protein coupled receptors. We here repor...
| Main Authors: | , , , , |
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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/1721.1/55362 |
| _version_ | 1826194222740406272 |
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| author | Khorana, H. Gobind Zhang, Shuguang Chelikani, Prashen Takayama, Hidehito Reeves, Philip J. |
| author2 | Massachusetts Institute of Technology. Center for Biomedical Engineering |
| author_facet | Massachusetts Institute of Technology. Center for Biomedical Engineering Khorana, H. Gobind Zhang, Shuguang Chelikani, Prashen Takayama, Hidehito Reeves, Philip J. |
| author_sort | Khorana, H. Gobind |
| collection | MIT |
| description | The study of membrane protein structure and function requires their high-level expression and purification in fully functional form. We previously used a tetracycline-inducible stable mammalian cell line, HEK293S-TetR, for regulated high-level expression of G-protein coupled receptors. We here report successfully using this method for high-level expression of de novo oligo-DNA assembled human CD81 gene. CD81 is a member of the vital tetraspanin membrane protein family. It has recently been identified as the putative receptor for the Hepatitis C Virus envelope E2 glycoprotein (HCV-E2). In this study we used a single-step rho-1D4-affinity purification method to obtain >95% purity from HEK293S-TetR-inducible stable cell lines. Using ELISA assay we determined that the affinity of the purified CD81 receptor for HCV-E2 protein is 3.8±1.2 nM. Using fluorescent confocal microscopy we showed that the inducibly overexpressed CD81 receptor in HEK293S-TetR cells is correctly located on the plasma membrane. We demonstrated that the combination of high-level expression of CD81 with efficient single-step immunoaffinity purification is a useful method for obtaining large quantities of CD81 membrane receptor suitable for detailed structural analyses of this elusive tetraspanin protein. Furthermore, this simple single-step immunoaffinity purification to high purity of membrane protein could be useful broadly for other membrane protein purifications, thus accelerating the determination of structures for large numbers of difficult-to-obtain membrane proteins. |
| first_indexed | 2024-09-23T09:52:47Z |
| format | Article |
| id | mit-1721.1/55362 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T09:52:47Z |
| publishDate | 2010 |
| publisher | Public Library of Science |
| record_format | dspace |
| spelling | mit-1721.1/553622022-09-26T14:15:50Z High-Level Expression, Single-Step Immunoaffinity Purification and Characterization of Human Tetraspanin Membrane Protein CD81 Khorana, H. Gobind Zhang, Shuguang Chelikani, Prashen Takayama, Hidehito Reeves, Philip J. Massachusetts Institute of Technology. Center for Biomedical Engineering Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemistry Khorana, H. Gobind Khorana, H. Gobind Zhang, Shuguang Chelikani, Prashen Takayama, Hidehito The study of membrane protein structure and function requires their high-level expression and purification in fully functional form. We previously used a tetracycline-inducible stable mammalian cell line, HEK293S-TetR, for regulated high-level expression of G-protein coupled receptors. We here report successfully using this method for high-level expression of de novo oligo-DNA assembled human CD81 gene. CD81 is a member of the vital tetraspanin membrane protein family. It has recently been identified as the putative receptor for the Hepatitis C Virus envelope E2 glycoprotein (HCV-E2). In this study we used a single-step rho-1D4-affinity purification method to obtain >95% purity from HEK293S-TetR-inducible stable cell lines. Using ELISA assay we determined that the affinity of the purified CD81 receptor for HCV-E2 protein is 3.8±1.2 nM. Using fluorescent confocal microscopy we showed that the inducibly overexpressed CD81 receptor in HEK293S-TetR cells is correctly located on the plasma membrane. We demonstrated that the combination of high-level expression of CD81 with efficient single-step immunoaffinity purification is a useful method for obtaining large quantities of CD81 membrane receptor suitable for detailed structural analyses of this elusive tetraspanin protein. Furthermore, this simple single-step immunoaffinity purification to high purity of membrane protein could be useful broadly for other membrane protein purifications, thus accelerating the determination of structures for large numbers of difficult-to-obtain membrane proteins. 2010-06-02T15:45:37Z 2010-06-02T15:45:37Z 2008-06 2008-03 Article http://purl.org/eprint/type/JournalArticle 1932-6203 http://hdl.handle.net/1721.1/55362 Takayama H, Chelikani P, Reeves PJ, Zhang S, Khorana HG (2008) High-Level Expression, Single-Step Immunoaffinity Purification and Characterization of Human Tetraspanin Membrane Protein CD81. PLoS ONE 3(6): e2314. doi:10.1371/journal.pone.0002314 en_US http://dx.doi.org/10.1371/journal.pone.0002314 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 | Khorana, H. Gobind Zhang, Shuguang Chelikani, Prashen Takayama, Hidehito Reeves, Philip J. High-Level Expression, Single-Step Immunoaffinity Purification and Characterization of Human Tetraspanin Membrane Protein CD81 |
| title | High-Level Expression, Single-Step Immunoaffinity Purification and Characterization of Human Tetraspanin Membrane Protein CD81 |
| title_full | High-Level Expression, Single-Step Immunoaffinity Purification and Characterization of Human Tetraspanin Membrane Protein CD81 |
| title_fullStr | High-Level Expression, Single-Step Immunoaffinity Purification and Characterization of Human Tetraspanin Membrane Protein CD81 |
| title_full_unstemmed | High-Level Expression, Single-Step Immunoaffinity Purification and Characterization of Human Tetraspanin Membrane Protein CD81 |
| title_short | High-Level Expression, Single-Step Immunoaffinity Purification and Characterization of Human Tetraspanin Membrane Protein CD81 |
| title_sort | high level expression single step immunoaffinity purification and characterization of human tetraspanin membrane protein cd81 |
| url | http://hdl.handle.net/1721.1/55362 |
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