High-Level Expression, Single-Step Immunoaffinity Purification and Characterization of Human Tetraspanin Membrane Protein CD81
Author(s)
Khorana, H. Gobind; Zhang, Shuguang; Chelikani, Prashen; Takayama, Hidehito; Reeves, Philip J.
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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.
Date issued
2008-06Department
Massachusetts Institute of Technology. Center for Biomedical Engineering; Massachusetts Institute of Technology. Department of Biology; Massachusetts Institute of Technology. Department of ChemistryJournal
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
Version: Final published version
ISSN
1932-6203