title: Dot-blot immunodetection as a versatile and high-throughput assay to evaluate recombinant GPCRs produced in the yeast Pichia pastoris.
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 beta2-adrenergic receptor expressed in P. pastoris as a benchmarking standard.
authors.affiliation: UMR7175 ESBS/CNRS, Department of Biotechnology of Molecular Interactions, Blvd.S. Brant, BP10413, 67412 Illkirch Cedex, France.
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Zeder-Lutz
authors.forename: Nadia
authors.initials: N
authors.lastname: Cherouati
authors.forename: Christoph
authors.initials: C
authors.lastname: Reinhart
authors.forename: Franc
authors.initials: F
authors.lastname: Pattus
authors.forename: Renaud
authors.initials: R
authors.lastname: Wagner
mesh_terms: D000327:Adsorption; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015151:Immunoblotting; D010843:Pichia / Q000235:genetics*; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance / Q000379:methods; D013499:Surface Properties; D013997:Time Factors
chemical_list: D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins