Structural genomics on membrane proteins: comparison of more than 100 GPCRs in 3 expression systems.
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.
Lundstrom
Kenneth
K
Bioxtal, Chemin des Croisettes 22, Epalinges CH-1066, Switzerland.
Wagner
Renaud
R
Reinhart
Christoph
C
Desmyter
Aline
A
Cherouati
Nadia
N
Magnin
Thierry
T
Zeder-Lutz
Gabrielle
G
Courtot
Melanie
M
Prual
Cécile
C
André
Nicolas
N
Hassaine
Gherici
G
Michel
Hartmut
H
Cambillau
Christian
C
Pattus
Franc
F
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
D008565:Membrane Proteins; D043562:Receptors, G-Protein-Coupled