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High-Level Expression, Single-Step Immunoaffinity Purification and Characterization of Human Tetraspanin Membrane Protein CD81

Takayama, Hidehito and Chelikani, Prashen and Reeves, Philip J and Zhang, Shuguang and Khorana, H Gobind (2008) 'High-Level Expression, Single-Step Immunoaffinity Purification and Characterization of Human Tetraspanin Membrane Protein CD81.' PLoS ONE, 3 (6). e2314-e2314. ISSN 1932-6203


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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, HEK2935-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 HEK2935-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 HEK2935 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. © 2008 Takayama et al.

Item Type: Article
Uncontrolled Keywords: Cell Line; Subcellular Fractions; Humans; Recombinant Proteins; Antigens, CD; Enzyme-Linked Immunosorbent Assay; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Cloning, Molecular; Protein Conformation; Glycosylation; Models, Molecular; Tetraspanin 28
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: Faculty of Science and Health
Faculty of Science and Health > Life Sciences, School of
SWORD Depositor: Elements
Depositing User: Elements
Date Deposited: 07 Oct 2011 14:16
Last Modified: 18 Aug 2022 11:13

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