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. 2006 May;15(5):1115-26.
doi: 10.1110/ps.062098206. Epub 2006 Apr 5.

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

Nicolas André  1 , Nadia Cherouati, Cécile Prual, Tania Steffan, Gabrielle Zeder-Lutz, Thierry Magnin, Franc Pattus, Hartmut Michel, Renaud Wagner, Christoph Reinhart
Affiliations

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

Nicolas André et al. Protein Sci. 2006 May.

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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Figures

Figure 1.
Figure 1.
Schematic drawing of the P. pastoris expression vector for heterologous production of 20 G protein-coupled receptors. (AOX1P) Alcohol oxidase 1 gene promoter, (α-F) coding region for the prepropeptide of the S. cerevisiae mating type factor α, (Flag) coding region for the FLAG-tag, (H10) His-tag consisting of 10 consecutive histidine codons, (TEV) coding region for the tobacco etch virus protease cleavage site, (Bio) coding region for the biotinylation domain of the transcarboxylase from Propionibacterium shermanii.
Figure 2.
Figure 2.
Radioligand binding studies on membranes of P. pastoris cells producing the following receptors: (A) ADA2B_HUMAN, (B) 5HT1B_HUMAN, (C) HRH2_HUMAN, (D) AA2A_HUMAN, (E) NK2R_RAT, (F) OPRK_HUMAN. Yeast cells were induced as described in Materials and Methods. Binding assays were performed under the conditions outlined in Table 1. Binding assays were performed as outlined in Materials and Methods. The results shown are from three independent experiments. (Solid line) Membranes from yeast cells grown under standard conditions, (broken line) membranes from yeast cells grown under optimized conditions.
Figure 3.
Figure 3.
Production level and its improvement factor for 20 GPCRs in various conditions. Cells were grown in BMMY at 30°C, 225 rpm, 22 h. For a, temperature was shifted to 20°C during induction; for b, BMMY medium was supplemented with 2.5% DMSO; for c, medium was supplemented with antagonist or agonist (100× Kd); for d, medium was supplemented with histidine (0.04 mg/mL). The binding values for standard expression conditions (Std. Expr.) and optimized expression conditions (Opt. Expr.) were calculated from three independent saturation curves. The improvement factor represents the Bmax value of the changed condition divided by the Bmax value of the standard conditions from a, b, c, or d. Bmax values from a, b, c, or d were calculated from single point measurements using 5–10× Kd for the radioactive ligand. For the optimized conditions, only parameters showing a positive effect (improvement factors >1) were combined.
Figure 4.
Figure 4.
Dot-blot experiments on yeast membranes producing the 20 GPCRs. (β2AR) Pichia membrane containing human β2 adrenergic receptor (25 pmol/mg), (1–20) receptor number according to Table 1, (C) control cells transformed with empty vector pPIC9K. One microgram (1 μg) of Pichia membrane was applied to PVDF membrane. The top row represents the results obtained from standard conditions; the bottom row, results from optimized conditions. Immunoblot analysis was performed using the anti-FLAG M2 antibody as described in Materials and Methods.
Figure 5.
Figure 5.
Dot-blot vs. ligand binding. Correlation between total and functional receptor. (Shaded bars) Improvement factor for total receptor production (dot-blot signal optimized condition divided by dot-blot signal standard conditions), (solid bars) improvement factor for functional receptor production (Bmax value optimized condition divided by Bmax value standard condition).
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