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Structural genomics on membrane proteins: comparison of more than 100 GPCRs in 3 expression systems

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Reinhart,  Christoph
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Prual,  Cécile
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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André,  Nicolas
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Michel,  Hartmut
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Citation

Lundstrom, K., Wagner, R., Reinhart, C., Desmyter, A., Cherouati, N., Magnin, T., et al. (2006). Structural genomics on membrane proteins: comparison of more than 100 GPCRs in 3 expression systems. Journal of Structural and Functional Genomics, 7(2), 77-91. doi:10.1007/s10969-006-9011-2.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0024-D9A4-7
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.