S. cerevisiae transformation and small-scale overexpression
The transformation and overexpression in S. cerevisiae were basically performed according to the previous method
. We here used the 2-μ vector pDDGFP-2
 and the vacuolar protease-deficient S. cerevisiae FGY217 (MATα, ura3-52, lys2Δ201, and pep4Δ)
. DNA fragments encoding the target GPCRs were amplified with the KOD Plus DNA polymerase (TOYOBO, Tokyo, Japan), with forward and reverse primers containing a 20–30 bp gene-specific region and a 30 bp homologous region
. Approximately 30 ng of pDDGFP-2 and 3 μL of 1 ~ 4 PCR fragments of a GPCR (which have a ~30 bp overlapping region with each other) were transformed. Transformants were selected on Ura- plates at 30 °C.
Colonies of transformants harboring the target GPCR were grown in 5 mL of Ura- medium with 2% glucose in 50 mL aerated capped tubes (TPP, Switzerland) at 30 °C overnight. The cultures were diluted to an OD600 of 0.12 and cultured in 10 mL of Ura- medium with 0.1% glucose at 30 °C. At an OD600 of 0.6, galactose was added to the culture to a final concentration of 2%, DMSO was added as needed, and the temperature was lowered to 20 °C as needed. After shaking for 20–22 h at 30 °C (or 40 h at 20 °C), the cells were harvested, and the cell pellets were resuspended in 700 μL of buffer A (50 mM Tris–HCl, pH 7.5, 5 mM EDTA, 10% glycerol, 0.12 M sorbitol, and complete protease inhibitor cocktail [Roche]). The cell suspensions were diluted 20-fold in buffer A, and whole-cell GFP fluorescence was measured with a SpectraMax M2e microplate reader (Molecular Devices, USA) in a 96-well black plate. Fluorescence at an emission wavelength of 525 nm was measured by using a 515 nm cutoff filter after excitation at 490 nm. Purified yEGFP was used as a standard for estimating overexpression.
Membranes from small-scale (10 mL) cultures were prepared as follows. A yeast cell suspension (700 μL) was transferred to 2 mL tubes containing 500 μL of acid-washed, dry, 425 to 600 μm glass beads (Sigma). Cells were disrupted on a Cutemixer CM-1000 (EYELA, Tokyo, Japan) at 2,500 rpm for 40 min at 4 °C. The samples were examined microscopically to confirm that >90% of the cells were broken. Unbroken cells and debris were pelleted by centrifugation, and the supernatant was transferred into an ultra-centrifuge tube. Yeast membranes were collected by ultracentrifugation at 100,000 g for 30 min at 4 °C. Prepared membranes were snap-frozen in liquid nitrogen and stored at −80 °C, or stored on ice and used within 24 h.
Intermediate and large-scale overexpression and membrane preparation in S. cerevisiae
Yeast clones harboring the target receptor were inoculated in Ura- medium with 2% glucose and grown at 30 °C overnight. The yeast culture was diluted to an OD600 of 0.12 in a total volume of 200 mL or more than 1 L of Ura- medium with 0.1% glucose in a 500 mL baffle flask or 2.5 L Tunair flask (Sigma-Aldrich, USA), respectively. The culture was shaken at 250 rpm at 30 °C, and expression was induced by adding galactose to a final concentration of 2% when the culture reached an OD600 of 0.6. After induction, the flasks were shaken for 20 h (30 °C cultures) or 40 h (20 °C cultures), and the cells were harvested. Cells were washed once with lysis buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 5% glycerol, complete protease inhibitor cocktail, and 2 mM EDTA), snap-frozen in liquid nitrogen, and stored at −80 °C.
The intermediate-scale cells were resuspended in the lysis buffer and disrupted in a 50 mL tube using an equal volume of glass beads on a Cutemixer at 2,500 rpm for 40 min at 4 °C. The large-scale cells were disrupted in a 2 L baffled flask using an equal volume of glass beads on an Innova 44R shaker (New Brunswick Scientific Inc., USA) for 60 min at 4 °C. Unbroken cells and debris were removed by centrifugation, the supernatant was transferred to ultracentrifuge tubes, and the membranes were pelleted by centrifugation at 100,000 g for 60 min at 4 °C. The pellet was suspended in buffer containing 50 mM Tris–HCl, pH 8.0, 120 mM NaCl, 20% glycerol and protease inhibitor cocktail, and stored at −80 °C.
Membranes containing each GPCR-GFP were uniformly resuspended by sonication in individual assay buffers ( Additional file
1: Table S2). Membrane proteins were quantified with the bicinchoninic acid (BCA) assay method (Pierce, USA). Membrane suspensions (5–20 μg) were incubated in triplicate with specific radioactively-labeled ligands ( Additional file
1: Table S2) for 1 h at 25 °C. Nonspecific binding was determined in the presence of excess unlabeled ligand. Membranes were trapped on Whatman GF/B filters that were presoaked in 0.3% polyethylenimine. The bound and free radioligands were separated by washing three times with water. The retained radioactivity was measured on an LCS-5100 liquid scintillation counter (ALOKA).
Fluorescence size exclusion chromatography (FSEC)
Membrane suspensions of GPCRs were solubilized in buffer containing 50 mM Tris–HCl, pH 7.5, 200 mM NaCl, protease inhibitor, and 1% detergent at a final concentration of 3 mg/mL total protein at 4 °C for 1 h with mild agitation. Insoluble material was pelleted by ultracentrifugation at 100,000 g at 4 °C for 30 min. FSEC was performed with a Superose 6 10/300 column (GE Healthcare) on a Biologic Chromatography System (BioRad) with 500 μL of solubilized sample. The column was preequilibrated with running buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% DDM/0.01% CHS). Fractions (0.2 mL) were collected in a 96-well microplate from the first 6 mL eluted after sample injection. Fluorescence emission at 525 nm was measured using a SpectraMax M2e plate reader with a 515 nm cutoff filter after excitation at 490 nm.
Overexpression in P. pastoris
Using the GPCR-integrated pDDGFP-2 plasmids as templates, coding regions of the GPCR-GFP fusion proteins were PCR-amplified with a forward primer containing a BamHI site (5′-CTA GAA CTA GTG GAT CCA CCA TG-3′) and a reverse primer containing an EcoRI site (5′-GCT TGA TAT CGA ATT CCT GCA GTT AAT G-3′). The PCR products were digested with BamHI and EcoRI and subcloned into the pPIC9K vector. The vector was linearized using PmeI. Transformation, clone selection, and small-scale culturing were performed as previously described
. The selected transformants were stored in glycerol stocks at −80 °C. Intermediate-scale (200 mL) and large-scale (more than 1 L) culturing were performed under the same conditions as small-scale culturing, with a 500 mL baffled flask and 2.5 L Tuniar Flask (Sigma-Aldrich, USA). P. pastoris cells from small- and intermediate-scale cultures were disrupted with glass beads and membranes were prepared in the same way as for S. cerevisiae.
Purification of GPCR variants
Purification of hHRH1-Nd-i3d expressed in P. pastoris and hHRH1-Nd-T4L expressed in S. cerevisiae was performed according to our previous report
. In brief, the membrane containing GPCR-GFP was solubilized by 1% (w/v) DDM/0.2% (w/v) CHS, and the unsolubilized material was separated by ultracentrifugation. The GPCR-GFP fusion protein was purified with TALON IMAC resin (Clontech). The purified protein was concentrated, and then the protein was treated overnight with His-tagged TEV protease (expressed and purified in house). TEV protease and the cleaved His-tagged GFP were removed from the sample by passing the sample through TALON resin and collecting the flow through. All the purification steps were performed in the presence of 100 μM pyrilamine.