Hassaine Gherici, Wagner Renaud, Kempf Juliette, Cherouati Nadia, Hassaine Nouzha, Prual Cecile, André Nicolas, Reinhart Christoph, Pattus Franc, Lundstrom Kenneth
BioXtal, Chemin des Croisettes 22, CH-1066 Epalinges, Switzerland.
Protein Expr Purif. 2006 Feb;45(2):343-51. doi: 10.1016/j.pep.2005.06.007. Epub 2005 Jul 11.
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.
用辛德毕斯病毒载体在三种哺乳动物细胞系中对101种G蛋白偶联受体进行评估。蛋白质免疫印迹法显示，101种受试GPCR中有95种显示出阳性信号。大量GPCR以高水平表达，表明受体产量在1 mg/L或更高范围内，适用于结构生物学应用。对选定数量的GPCR进行特异性结合试验，以比较总蛋白表达与功能蛋白表达之间的相关性。评估添加到细胞培养基中的配体和添加剂对表达增强的作用。还从突变型SFV载体表达选定的GPCR，以提高蛋白质表达并降低宿主细胞毒性，从而进一步提高受体产量。
