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 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.
Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
Hidehito
H
Takayama
Prashen
P
Chelikani
Philip J
PJ
Reeves
Shuguang
S
Zhang
H Gobind
HG
Khorana
D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions / Q000276:immunology; D060225:Tetraspanin 28
D015703:Antigens, CD; C554798:CD81 protein, human; D011994:Recombinant Proteins; D060225:Tetraspanin 28