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 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.
André
Nicolas
N
Max-Planck-Institute of Biophysics, D-60438 Frankfurt am Main, Germany.
Cherouati
Nadia
N
Prual
Cécile
C
Steffan
Tania
T
Zeder-Lutz
Gabrielle
G
Magnin
Thierry
T
Pattus
Franc
F
Michel
Hartmut
H
Wagner
Renaud
R
Reinhart
Christoph
C
D000595:Amino Acid Sequence; D002352:Carrier Proteins; D003001:Cloning, Molecular / Q000379:methods*; D015870:Gene Expression; D015151:Immunoblotting; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D010843:Pichia / Q000737:chemistry*; D011485:Protein Binding; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D046509:Tissue Culture Techniques
D002352:Carrier Proteins; D008024:Ligands; D043562:Receptors, G-Protein-Coupled