Recent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. Therefore, the quick screening of functionally expressed stable receptor variants is vital.
We developed a platform using Saccharomyces cerevisiae for the rapid construction and evaluation of functional GPCR variants for structural studies. This platform enables us to perform a screening cycle from construction to evaluation of variants within 6–7 days. We firstly confirmed the functional expression of 25 full-length class A GPCRs in this platform. Then, in order to improve the expression level and stability, we generated and evaluated the variants of the four GPCRs (hADRB2, hCHRM2, hHRH1 and hNTSR1). These stabilized receptor variants improved both functional activity and monodispersity. Finally, the expression level of the stabilized hHRH1 in Pichia pastoris was improved up to 65 pmol/mg from negligible expression of the functional full-length receptor in S. cerevisiae at first screening. The stabilized hHRH1 was able to be purified for use in crystallization trials.
We demonstrated that the S. cerevisiae system should serve as an easy-to-handle and rapid platform for the construction and evaluation of GPCR variants. This platform can be a powerful prescreening method to identify a suitable GPCR variant for crystallography.
Keywords:G-protein coupled receptor; Membrane protein; High expression; Screening; Receptor variants; Structural study; Saccharomyces cerevisiae
G-proteincoupled receptors (GPCRs), which represent the largest family of integral membrane proteins, play pivotal roles in mediating signal transduction events in response to ligands such as peptides and amines. GPCRs are major therapeutic drug targets and represent ~ 30% of the market share of all prescription drugs . Although the high-resolution 3D structures of the target GPCRs provide good initial models for drug design, difficulties in expression and purification have been a major bottleneck for structural study. Large quantities of high-quality pure protein are generally required for X-ray crystallography. With the exception of rhodopsin [2-4], which is naturally abundant and can be isolated from rod outer membranes in the eyes, GPCRs generally are not sufficiently abundant to be isolated from their endogenous tissues. Therefore, overexpression in a heterologous host is needed. Various types of hosts have been evaluated for use in GPCR expression, including bacteria, yeast, insect, and mammalian cells, as well as cell-free systems [5,6]. However, only a limited number of GPCRs have been successfully expressed and purified on a large scale. One reason for that may be their instability, which is most likely due to their dynamic activity in the membrane. Recent successes in structure determination have demonstrated the importance of stabilizing receptor in order to achieve high expression and/or facilitation of crystallization [7-13].
Because it is almost impossible to predict what modifications will improve the expression and/or stability of receptors, suitable variants must be selected from the pool of possible variants by trial and error. To facilitate structural studies of GPCRs, a screening system is required that will enable rapid selection of variants. Insect cells have been used as a successful host for structural study, but the screening is laborious and time consuming. E. coli has recently been used to screen the thermally stable GPCR variants of turkey β1 adrenergic receptor (tADRB1) , human adenosine A2a receptor (hADORA2A) [15,16], and rat neurotensin receptor 1 (rNTSR1) . In addition the crystal structures of the stabilized variants were determined for tADRB1 and hADORA2A [18,19]. However, only a limited number of functionally expressed receptors have been successfully generated in E. coli.
Yeast is a more preferred host for the expression of GPCRs than E. coli. Yeast has a protein quality control system similar to that of mammalian cells, which enables numerous posttranslational modifications and correct disulfide formation of mammalian membrane proteins. This similarity may lead to more functional expression of GPCRs in yeast . S. cerevisiae in particular is stable for protein expression, easy to manipulate, and quick to proliferate. S. cerevisiae has been extensively tailored for the screening of functional GPCR mutants . In addition, many GPCRs can be as highly expressed in yeast as in mammalian cells [21,23].
We previously established a GFP-based pipeline for the expression and purification of non-GPCR membrane proteins in S. cerevisiae[24,25]. S. cerevisiae permits the rapid cloning of genes of interest into the 2-μ plasmid by homologous recombination, enabling the direct expression and evaluation of the proteins. The amount and integrity of the target membrane protein can be estimated from the whole-cell fluorescence and in-gel fluorescence after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Monodispersity, which is a good indicator for purification, can be observed by fluorescence-detection size exclusion chromatography (FSEC) . The gene of a target protein can be transformed with the divided PCR fragments in one step . In the present study, we demonstrate that the platform using S. cerevisiae is very useful for the rapid construction and evaluation of GPCR variants for structural study. The stabilized GPCRs in S. cerevisiae were expressed at higher levels in P. pastoris yeast. Finally, the stabilized human histamine H1 receptor was successfully purified for structural biology study.
GFP-based platform for the rapid construction and evaluation of GPCR variants in S. cerevisiae
The GFP-based platform using S. cerevisiae for the construction and evaluation of GPCR variants is illustrated in Figure 1. The GPCR variants were designed and the genes were generated as PCR fragments (Figure 1A). The 2-μ plasmid named pDDGFP-2, which has a GAL1 promoter, and S. cerevisiae strain FGY217 were used  (Figure 1B). This plasmid/strain combination resulted in the best expression of membrane proteins. The genes of interest were integrated into the plasmid by homologous recombination in S. cerevisiae in one step via introduction of a mixture of linearized plasmid and PCR products. The clone harboring the GPCR variant is selected on an agar plate without uracil (Ura-). After small-scale (10 mL) culturing, the functional expressions are evaluated by radioligand-binding assays. The monodispersity of the detergent-solubilized receptor is assessed by FSEC by detecting the C-terminal GFP, which enables the evaluation without purification. In the present study, the SEC eluate was collected in a 96-well microplate and the fluorescence was detected by a plate reader following the existing protocol [24,25]. Therefore, a relatively large amount of samples (2 ~ 3 mg of total membrane protein) was needed, and the membranes were prepared from an intermediate-scale (200 mL) culture. We have confirmed that a similar result could be obtained from a small-scale (10 mL) culture by using a fluorescence detector at the outlet of the SEC column (data not shown). By omitting the intermediate-scale culturing, this platform enables us to perform a screening cycle within 6–7 days, compared to 16–18 days in P. pastoris or 30–35 days in insect cells using baculovirus (Figure 1C).
Figure 1. Overview of the construction and evaluation platform of GPCR variants in S. cerevisiae. (A) Primer design of a variant. As an example, the hHRH1 variant with truncation of the N-terminal region, a mutation at the 3.41 position in TM3 and T4L fusion to the i3-loop is shown. The four PCR fragments are generated using the indicated primer pairs from full-length GPCR and T4L ( Additional file 1: Table S1). The same colored overlapping regions were necessary for homologous recombination in S. cerevisiae. (B) Illustration of a cycle from construction design to evaluation for the GPCR variants. (C) Flow and time-scale of the construction and evaluation of GPCR variants in three hosts. Schemes for small-scale culturing are shown in yellow and orange, respectively. Evaluations by ligand-binding assays and FSEC are shown in cyan and green, respectively. Vec.: preparation of expression vector, Bac.: preparation of Bacmid, Ura-: selection on a Ura- plate, MD: selection on a minimum dextrose (MD) plate, Gen: selection on a geneticine plate, P1 and P2: P1 and P2 virus preparation, respectively.
Additional file 1. Supplementary information. Table S1 Primers used for the construction of hHRH1 variant (Nd-F116W-T4L). Table S2 Ligands and conditions for the single point radioligand binding assays. Figure S1 In-gel fluorescence of 25 GPCR-GFP fusions expressed in S. cerevisiae. Arrowheads represent the GPCR-GFP fusion bands and the asterisk represents an endogenous fluorescent ‘background’ protein from S. cerevisiae that migrates at approximately 70 kDa. Figure S2 Construction design of GPCR variants. (A) Sequence alignments of transmembrane 3 (TM3) of the GPCRs in this study with bovine rhodopsin and human ADORA2A. The number above the sequence is the general indexed position based on the Ballesteros–Weinstein system. The 3.41 position for receptor stabilization is highlighted in yellow. (B) Sequence alignments of TM5, i3-loop, and TM6. The position where the T4 lysozyme sequence is fused is shown in red. To truncate the long i3 loop, the residues shown in blue were connected for each receptor. Figure S3 Fluorescence intensity and activity of GPCR variants screened in S. cerevisiae. Whole-cell GFP fluorescence (arbitrary unit, bar graph) and specific activity of the membrane by radioligand binding assays (black square plot) of full-length GPCRs and GPCR variants constructed in S. cerevisiae. (A) hADRB2, (B) hCHRM2, (C) hHRH1, and (D) hNTSR1. Figure S4 Evaluation of the GPCR variants expressed in Sf9 insect cells. The specific binding activities (left) and FSEC profiles (right) of full-length GPCRs and the improved GPCR variants expressed in Sf9 cells are shown. The colors of the chromatogram correspond to those in the binding assays. (A) hADRB2, (B) hCHRM2, (C) hHRH1, (D) hNTSR1. FSEC was performed with a Superose 6 10/300 column. The void peak is denoted by an asterisk. The arrow indicates the target peak of GPCR fused to GFP.
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Overexpression of full-length GPCRs in S. cerevisiae
First, 25 full-length GPCRs were expressed and evaluated using this platform. The integrity of the GPCR-GFP fusions examined by in-gel fluorescence after standard SDS-PAGE  indicated that most of the GPCRs were not degraded and appeared as a single major band in the gel ( Additional file 1: Figure S1). Table 1 shows the expression levels of full-length receptors estimated by the GFP fluorescence (total expression) and single-point radioligand-binding assay (functional expression). Without any signal sequence and under standard culture conditions (no additives and at 30 °C), the ligand-binding activities of 12 GPCRs were zero or lower than 0.1 pmol/mg. The N-terminal yeast alpha mating factor signal sequence which improved the ligand-binding activities of GPCRs [29-32]. In the previous report on GPCR expression in P. pastoris, supplementation of dimethyl sulfoxide (DMSO) and lowering induction temperature to 20 °C increased the ligand-binding activity of receptor . Then four culture conditions were examined: the presence or absence of 2.5% DMSO and two induction temperatures (20 °C, 30 °C). The functional expression of 24 of 25 GPCRs was increased in S. cerevisiae in the optimized conditions. For many receptors, there was little correlation between the intensity of GFP fluorescence and ligand-binding activity, suggesting that the intensity of GFP fluorescence itself is not a good indicator of functional expression in this platform. After these optimizations, 25 GPCRs were functionally expressed. However, the expression level remained insufficient for structural study.
Table 1. Summary of the expression level and functional activity of the 25 GPCRs expressed S. cerevisiae under different culture conditions in small-scale cultures
Use of S. cerevisiae as a platform for the construction and evaluation of GPCR variants
We generated and evaluated the variants of the three GPCRs shown in Table 2 (hCHRM2, hHRH1, hNTSR1) using the GFP-based platform in S. cerevisiae. In addition, the hADRB2 variant (E122W-N187E-Cd), whose structure has been solved as a T4L-fusion , was also generated. To construct GPCR variants, the following four variant modules were considered: (1) A truncation of flexible long N- (Nd) or C-terminal residues (Cd), which effectively increases GPCR expression in some reported cases (e.g., ref. ). (2) A point mutation at the 3.41 position in transmembrane helix 3 ( Additional file 1: Figure S2A). The numbering is based on the general indexed position in the Ballesteros-Weinstein system . Mutations at this position reportedly increase the thermal stability of hADRB2 . (3) A deletion mutant of a long third intracellular loop (i3-loop) (abbreviated as i3d; Additional file 1: Figure S2B). A long i3-loop potentially becomes a target of degradation or receptor destabilization on the host cell surface. For muscarinic receptors, deletion of the i3-loop results in higher functional expression [38,39]. (4) Replacement of part of the i3-loop by T4 lysozyme (T4L, residues from 2 to 161) ( Additional file 1: Figure S2B); this replacement has been successful in crystallization and structural determination of hADRB2, hADORA2A, hDRD3 and hCXCR4 in cubic phase crystals [8,11-13].
Table 2. GPCR variants constructed and evaluated in S. cerevisiae in this study
After small-scale culturing, the total receptor expressions and functional expressions were determined by GFP fluorescence and radioligand-binding assay, respectively ( Additional file 1: Figure S3). These results showed that the intensity of GFP fluorescence itself is not a good indicator of functional expression. The ligand binding activity and monodispersity of the detergent-solubilized receptor of the variants that portrayed improved functional expression are shown in Figure 2. The hADRB2 variant Cd-E122W-N187E showed a ~ 10-fold increase in ligand-binding activity. The hCHRM2 variant Nd-i3d showed a 2.5-fold increase. The hHRH1 variants Nd-i3d and Nd-T4L displayed 7- and 26-fold increased activity, respectively. In hNTSR1, mutagenesis at the 3.41-position (Nd-L157W-Cd and Nd-L157W-T4L-Cd) increased activity by ~ 1.5-fold. Importantly, the variants that showed improved activity also exhibited improved FSEC profiles after solubilization with the mixed micelle of n-Dodecyl-β-D-maltopyranoside (DDM) and cholesteryl hemisuccinate (CHS), which is commonly used for purification of GPCRs. This indicates that there is a correlation between the improvement in both ligand-binding activity and monodispersity in GPCR variants.
Figure 2. Specific binding activity (left) and FSEC profile (right) of the full-length and improved variants expressed in S. cerevisiae. (A) hADRB2, (B) hCHRM2, (C) hHRH1, and (D) hNTSR1. FSEC was performed with the Superose 6 10/300 column. The colors of the FSEC chromatogram correspond to those of the binding activity. The void peak is denoted by an asterisk. An arrow indicates the target peak of GPCR-GFP fusion.
Expression of the GPCR variants in other hosts
Human HRH1-Nd-T4L variant showed a high expression (16 pmol/mg) and good FSEC profile in S. cerevisiae. However, the expression of the other GPCR variants remained too low for purification. If the improved variants could be expressed in another host at higher levels, purification would be facilitated. While insect cells are currently known as the most successful host for GPCR expression for structural study, our recent successes in the structural determination of hHRH1 and human adenosine A2a receptor (hADORA2a) were achieved by using P. pastoris as a host cell [40,41]. P. pastoris is easier to handle compared to insect cells, and can generate milligram quantities of high-quality mammalian GPCR protein as well as insect cells [42-46]. Therefore, we attempted to express selected GPCR variants in P. pastoris.
Improvements of ligand-binding activity and FSEC profiles of GPCR variants were also observed in P. pastoris (Figure 3). The hADRB2 variant E122W-N187E-Cd exhibited a large improvement in ligand-binding activity (80-fold) and a larger peak derived from monodisperse receptor in P. pastoris. In the hCHRM2 variant Nd-i3d, improvements in both ligand-binding activity (2-fold) and monodispersity were observed. For hHRH1, substantial improvements in ligand-binding activity and monodispersity in FSEC were observed for both Nd-i3d and Nd-T4L (by 6- and 4-fold, respectively). Although the improvements in ligand-binding activity of the hNTSR1 variants Nd-157W-Cd and Nd-157W-T4L-Cd were only ~ 1.5-fold in S. cerevisiae, ligand-binding activity and monodispersity were largely improved in P. pastoris. We confirmed that improvements of GPCR variants were also observed in Sf9 insect cells ( Additional file 1: Figure S4). For the receptors in the present study, the expression level in P. pastoris was the same or higher than that in insect cells.
Figure 3. Evaluation of the GPCR variants expressed in P. pastoris. The specific binding activities (left) and FSEC profiles (right) of full-length GPCRs and the improved GPCR variants expressed in P. pastoris are shown. The colors of the chromatogram correspond to those in the binding assays. (A) hADRB2, (B) hCHRM2, (C) hHRH1, (D) hNTSR1. FSEC was performed with a Superose 6 10/300 column. The void peak is denoted by an asterisk. The arrow indicates the target peak of GPCR fused to GFP.
Purification of the GPCR variants
The variants, hHRH1-Nd-i3d expressed in P. pastoris and hHRH1-Nd-T4L expressed in S. cerevisiae, were successfully purified in milligram quantities from a large-scale culture. The yield of functional expression estimated from ligand binding assay were more than 0.3 mg per 1 L culture for hHRH1-Nd-i3d expressed in P. pastoris and more than 0.04 mg for hHRH1-Nd-T4L expressed in S. cerevisiae. The yield estimated from whole-cell GFP fluorescence intensities were ~5 mg for hHRH1-Nd-i3d expressed in P. pastoris and ~1.2 mg for hHRH1-Nd-T4L expressed in S. cerevisiae. The final yield after purification of hHRH1-Nd-i3d expressed in P. pastoris, hHRH1-Nd-T4L expressed in S. cerevisiae were 0.3 ~ 0.4 mg and 0.04 mg per 1 L culture, respectively. These purified GPCR variants showed 90 ~ 95% purity judging from SDS-PAGE and showed a high degree of monodispersity judging from size exclusion chromatography (SEC) with absorbance detection at 280 nm (Figure 4). The SEC profile of purified hHRH1-Nd-T4L expressed in P. pastoris was also monodisperse as described in our recent report .
Figure 4. Purification of the receptor variant. (A) hHRH1-Nd-i3d expressed in P. pastoris, and (B) hHRH1-Nd-T4L expressed in S. cerevisiae. The size-exclusion chromatogram using Superdex 200 10/300 is shown in the left panel, and the Coomassie blue-stained SDS-PAGE gel is shown in the right panel. Lane 1, molecular weight marker. Lane 2, purified receptor variant. Arrows indicate the purified receptors.