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

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 DMS...

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Bibliographic Details
Published in:Protein science 2006-05, Vol.15 (5), p.1115-1126
Main Authors: André, Nicolas, Cherouati, Nadia, Prual, Cécile, Steffan, Tania, Zeder‐Lutz, Gabrielle, Magnin, Thierry, Pattus, Franc, Michel, Hartmut, Wagner, Renaud, Reinhart, Christoph
Format: Article
Language:English
Subjects:
Amino Acid Sequence
binding assay
Carrier Proteins
Cloning, Molecular - methods
expression screening
G protein‐coupled receptor
Gene Expression
GPCR
Immunoblotting
Kinetics
Ligands
Molecular Sequence Data
optimization
Pichia - chemistry
Pichia pastoris
Protein Binding
Radioligand Assay
Receptors, G-Protein-Coupled - analysis
Receptors, G-Protein-Coupled - chemistry
Receptors, G-Protein-Coupled - isolation & purification
Tissue Culture Techniques
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Summary: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.
ISSN:0961-8368
1469-896X
DOI:10.1110/ps.062098206