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...

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Main Authors: Khorana, H. Gobind (Contributor), Zhang, Shuguang (Contributor), Chelikani, Prashen (Contributor), Takayama, Hidehito (Contributor), Reeves, Philip J. (Author)
Other Authors: Massachusetts Institute of Technology. Center for Biomedical Engineering (Contributor), Massachusetts Institute of Technology. Department of Biology (Contributor), Massachusetts Institute of Technology. Department of Chemistry (Contributor)
Format: Article
Language:English
Published: Public Library of Science, 2010-06-02T15:45:37Z.
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Online Access:Get fulltext
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100 1 0 |a Khorana, H. Gobind  |e author 
100 1 0 |a Massachusetts Institute of Technology. Center for Biomedical Engineering  |e contributor 
100 1 0 |a Massachusetts Institute of Technology. Department of Biology  |e contributor 
100 1 0 |a Massachusetts Institute of Technology. Department of Chemistry  |e contributor 
100 1 0 |a Khorana, H. Gobind  |e contributor 
100 1 0 |a Khorana, H. Gobind  |e contributor 
100 1 0 |a Zhang, Shuguang  |e contributor 
100 1 0 |a Chelikani, Prashen  |e contributor 
100 1 0 |a Takayama, Hidehito  |e contributor 
700 1 0 |a Zhang, Shuguang  |e author 
700 1 0 |a Chelikani, Prashen  |e author 
700 1 0 |a Takayama, Hidehito  |e author 
700 1 0 |a Reeves, Philip J.  |e author 
245 0 0 |a High-Level Expression, Single-Step Immunoaffinity Purification and Characterization of Human Tetraspanin Membrane Protein CD81 
260 |b Public Library of Science,   |c 2010-06-02T15:45:37Z. 
856 |z Get fulltext  |u http://hdl.handle.net/1721.1/55362 
520 |a 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. 
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655 7 |a Article 
773 |t PLoS ONE