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

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.

Expression, purification and characterization of a phyA m -phyCs fusion phytase

“…This is the first report of a fusion phytase gene in P. pastoris which has been shown to be suitable for high-level expression of various heterologous proteins, either intracellular or secretory. Under fermentation conditions P. pastoris has been shown to be able to grow to high cell densities capable of giving high levels of expressed protein with levels greater than 10 g/L reported (Cregg and Higginns, 1995;Romanos, 1995;André et al, 2006). So there is a huge potential for the fermentation of fusion phytase if optimizing the cultivation conditions.…”

GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

“…Note that, for the initial optimization, we recommend adding either DMSO or His, as we find that the presence of these chemical chaperones can afford an additional 30% improvement to the whole-cell expression levels 6 . Until now it was unclear as to why these chemical chaperones can improve membrane protein production levels 20 .…”

“…Using Steps 20-23, one can also test other culture conditions that have shown to be successful to improve membrane protein production, for example, the addition of a ligand to the culture medium 20 , or to switch the culture to 20 °C after galactose induction. However, in the latter case, we have found that only 5% of the targets we have tested give an additional improvement in expression compared with standard growth at 30 °C (ref.…”

“…Repeat the membrane protein overexpression culturing condition that gave the highest level of whole-cell fluorescence (Steps [20][21][22]. Resuspend harvested cells in 1 ml of -URA media containing 50% glycerol.…”

Single-cell profiling reveals GPCR heterogeneity and functional patterning during neuroinflammation

“…However, due to the limited amount of RNA obtained from an individual cell, it cannot be excluded that -due to the technical limitations of single-cell expression analysis -very low abundance transcripts remain undetected, in particular if the RNA quality is compromised. Since GPCRs are, in many cases, expressed at very low levels (43), it is in this context important to consider the sensitivity of the detection system used for single-cell expression analysis. A recent study directly compared the 2 major readout systems for single-cell expression analysis, RT-PCR and RNA sequencing (RNA-Seq), with respect to their sensitivity for GPCR detection in murine aortic smooth muscle cells.…”