High-Level Expression, Single-Step Immunoaffinity Purification and Characterization of Human Tetraspanin Membrane Protein CD81
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Takayama-2008-High-Level Expressio.pdf
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Author(s)
Khorana, H. Gobind
Zhang, Shuguang
Chelikani, Prashen
Takayama, Hidehito
Reeves, Philip J.
Date Issued
June 2008
Journal
PLoS ONE
Publisher
Public Library of Science
Citation
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
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Final published version
Abstract
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.
MIT Department
Massachusetts Institute of Technology. Center for Biomedical Engineering
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Chemistry
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http://dx.doi.org/10.1371/journal.pone.0002314
