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  Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen.

André, N., Cherouati, N., Prual, C., Steffan, T., Zeder-Lutz, G., Magnin, T., et al. (2006). Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen. Protein Science, 15(5), 1115-1126. doi:10.1110/ps.062098206.

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 Creators:
André, Nicolas1, Author              
Cherouati, Nadia2, Author
Prual, Cécile1, Author              
Steffan, Tanja2, Author
Zeder-Lutz, Gabrielle2, Author
Magnin, Thierry2, Author
Pattus, Franc2, Author
Michel, Hartmut1, Author              
Wagner, Renaud2, Author
Reinhart, Christoph1, Author              
Affiliations:
1Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              
2Unité Mixte de Recherche (UMR)7100 Ecole Supérieur de Biotechnologie de Strasbourg-Centre National de la Recherche (ESBS-CNRS), Department Récepteurs et Protéines Membranaires, 67412 Illkirch, France, ou_persistent22              

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Free keywords: G protein‐coupled receptor; GPCR; expression screening; binding assay; optimization
 Abstract: We have optimized the expression level of 20 mammalian G protein-coupled receptors (GPCRs) in the methylotrophic yeast Pichia pastoris. We found that altering expression parameters, including growth temperature, and supplementation of the culture medium with specific GPCR ligands, histidine, and DMSO increased the amount of functional receptor, as assessed by ligand binding, by more than eightfold over standard expression conditions. Unexpectedly, we found that the overall amount of GPCR proteins expressed, in most cases, varied only marginally between standard and optimized expression conditions. Accordingly, the optimized expression conditions resulted in a marked fractional increase in the ratio of ligand binding-competent receptor to total expressed receptor. The results of this study suggest a general approach for increasing yields of functional mammalian GPCRs severalfold over standard expression conditions by using a set of optimized expression condition parameters that we have characterized for the Pichia expression system. Overall, we have more than doubled the number of GPCR targets that can be produced in our laboratories in sufficient amounts for structural studies.

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Language(s): eng - English
 Dates: 2006-02-072006-01-182006-02-072009-01-012006-05
 Publication Status: Published in print
 Pages: 12
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1110/ps.062098206
PMID: 16597836
 Degree: -

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Title: Protein Science
Source Genre: Journal
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Publ. Info: New York, N.Y. : Cambridge University Press
Pages: - Volume / Issue: 15 (5) Sequence Number: - Start / End Page: 1115 - 1126 Identifier: ISSN: 0961-8368
CoNE: https://pure.mpg.de/cone/journals/resource/954925342760