Semliki Forest virus vectors for overexpression of 101 G protein-coupled receptors in mammalian host cells

• Published in: Protein expression and purification Vol. 45; no. 2; pp. 343 - 351
• Main Authors: Hassaine, Gherici, Wagner, Renaud, Kempf, Juliette, Cherouati, Nadia, Hassaine, Nouzha, Prual, Cecile, André, Nicolas, Reinhart, Christoph, Pattus, Franc, Lundstrom, Kenneth
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2006; Elsevier
• Subjects: Animals; Binding assays; Biotechnology; Cell Line; Cricetinae; Culture Media - chemistry; G protein-coupled receptors; Gene Expression Profiling; Genetic Vectors - genetics; Genetic Vectors - metabolism; Humans; Immunodetection; Large-scale production; Life Sciences; Mammalian cell lines; Molecular Sequence Data; Radioligand Assay; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Recombinant expression; Semliki Forest virus; Semliki forest virus - genetics; Semliki forest virus - metabolism; Structural biology; Structural genomics; Recombinant expression; G protein-coupled receptors; Semliki Forest virus; Immunodetection; Large-scale production; Mammalian cell lines; Binding assays; Structural genomics; Structural biology
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2005.06.007

Semliki Forest virus vectors were applied for the evaluation of 101 G protein-coupled receptors in three mammalian cell lines. Western blotting demonstrated that 95 of the 101 tested GPCRs showed positive signals. A large number of the GPCRs were expressed at high levels suggesting receptor yields in the range of 1 mg/L or higher, suitable for structural biology applications. Specific binding assays on a selected number of GPCRs were carried out to compare the correlation between total and functional protein expression. Ligands and additives supplemented to the cell culture medium were evaluated for expression enhancement. Selected GPCRs were also expressed from mutant SFV vectors providing enhanced protein expression and reduced host cell toxicity in attempts to further improve receptor yields.