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Dot-blot; G-protein-coupled receptors; Pichia pastoris; Solubilization
Abstract:
One of the major objectives of the Membrane Protein Network program (MePNet, www.mepnet.org) is to express one hundred G-protein-coupled receptors (GPCRs) in the yeast Pichia pastoris. We have developed an antibody-based assay in order to select for the best behaving clones at each step of the receptor preparation, from expression to solubilization. This assay allowed us to quantify the expression of Flag-tagged GPCRs present in various sample types, from crude P. pastoris extracts to native membrane preparations and detergent solubilised fractions. It combines the specificity of ELISA, the sensitivity of enhanced chemiluminescence detection and the speed of high-throughput screening, and can detect as low as 0.001% (w/w) flag-tagged recombinant receptor present in a crude extract. The method was applied to sort recombinant GPCR clones, to rank receptor expression levels and to screen for detergent solubilization efficiency using the human β2-adrenergic receptor expressed in P. pastoris as a benchmarking standard.
