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.
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Acknowledgements
We are grateful for the technical assistance provided by Juliette Kempf and Tania Steffen (UMR7175, Illkirch, France), Gabriele Maul (Max Planck Institute of Biophysics, Frankfurt, Germany), Marie-Eve Gravière and Céline Huyghe (University of Marseille, France) and Mrs. Nouzha Hassaine (BioXtal, Epalinges, Switzerland). We are also thankful for the help with E. coli scale-up and fermentation from Prof. Rainer Rudolph and Mr. Jan Oschman (Martin-Luther University, Halle, Germany). We would like to thank all those academic and industrial contributors who provided us with cDNAs for several GPCRs used in this study. The financial support received for the MePNet program from the industrial partners is acknowledged.
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Lundstrom, K., Wagner, R., Reinhart, C. et al. Structural genomics on membrane proteins: comparison of more than 100 GPCRs in 3 expression systems. J Struct Funct Genomics 7, 77–91 (2006). https://doi.org/10.1007/s10969-006-9011-2
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DOI: https://doi.org/10.1007/s10969-006-9011-2