title: Structural genomics on membrane proteins: comparison of more than 100 GPCRs in 3 expression systems.
abstract: Production of recombinant receptors has been one of the major bottlenecks in structural biology on G protein-coupled receptors (GPCRs). The MePNet (Membrane Protein Network) was established to overexpress a large number of GPCRs in three major expression systems, based on Escherichia coli, Pichia pastoris and Semliki Forest virus (SFV) vectors. Evaluation by immunodetection demonstrated that 50% of a total of 103 GPCRs were expressed in bacterial inclusion bodies, 94% in yeast cell membranes and 95% in SFV-infected mammalian cells. The expression levels varied from low to high and the various GPCR families and subtypes were analyzed for their expressability in each expression system. More than 60% of the GPCRs were expressed at milligram levels or higher in one or several systems, compatible to structural biology applications. Functional activity was determined by binding assays in yeast and mammalian cells and the correlation between immunodetection and binding activity was analyzed.
authors.affiliation: Bioxtal, Chemin des Croisettes 22, Epalinges CH-1066, Switzerland.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Lundstrom
authors.forename: Renaud
authors.initials: R
authors.lastname: Wagner
authors.forename: Christoph
authors.initials: C
authors.lastname: Reinhart
authors.forename: Aline
authors.initials: A
authors.lastname: Desmyter
authors.forename: Nadia
authors.initials: N
authors.lastname: Cherouati
authors.forename: Thierry
authors.initials: T
authors.lastname: Magnin
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Zeder-Lutz
authors.forename: Melanie
authors.initials: M
authors.lastname: Courtot
authors.forename: Cécile
authors.initials: C
authors.lastname: Prual
authors.forename: Nicolas
authors.initials: N
authors.lastname: André
authors.forename: Gherici
authors.initials: G
authors.lastname: Hassaine
authors.forename: Hartmut
authors.initials: H
authors.lastname: Michel
authors.forename: Christian
authors.initials: C
authors.lastname: Cambillau
authors.forename: Franc
authors.initials: F
authors.lastname: Pattus
mesh_terms: D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005800:Genes, Fungal; D005814:Genes, Viral; D005822:Genetic Vectors; D023281:Genomics; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D011485:Protein Binding; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012672:Semliki forest virus / Q000235:genetics
chemical_list: D008565:Membrane Proteins; D043562:Receptors, G-Protein-Coupled