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.
Subject(s)
Escherichia coli , Gene Expression , Genomics , Membrane Proteins , Pichia , Receptors, G-Protein-Coupled , Semliki forest virus , Animals , Blotting, Western , Cloning, Molecular , Escherichia coli/genetics , Genes, Bacterial , Genes, Fungal , Genes, Viral , Genetic Vectors , Humans , Immunoblotting , Membrane Proteins/biosynthesis , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Pichia/genetics , Protein Binding , Radioligand Assay , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Semliki forest virus/geneticsABSTRACT
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.
Subject(s)
Cloning, Molecular/methods , Pichia/chemistry , Receptors, G-Protein-Coupled/chemistry , Amino Acid Sequence , Carrier Proteins , Gene Expression , Immunoblotting , Kinetics , Ligands , Molecular Sequence Data , Protein Binding , Radioligand Assay , Receptors, G-Protein-Coupled/analysis , Receptors, G-Protein-Coupled/isolation & purification , Tissue Culture TechniquesABSTRACT
Semliki Forest virus vectors were applied for the evaluation of 101 G protein-coupled receptors in three mammalian cell lines. Western blotting demonstrated that 95 of the 101 tested GPCRs showed positive signals. A large number of the GPCRs were expressed at high levels suggesting receptor yields in the range of 1 mg/L or higher, suitable for structural biology applications. Specific binding assays on a selected number of GPCRs were carried out to compare the correlation between total and functional protein expression. Ligands and additives supplemented to the cell culture medium were evaluated for expression enhancement. Selected GPCRs were also expressed from mutant SFV vectors providing enhanced protein expression and reduced host cell toxicity in attempts to further improve receptor yields.
