Baculoviruses were prepared using the Bac-to-Bac Baculovirus Expression System (Thermo Fisher). Sf9 insect cells were cultured to a density of 3 × 106 cells per mL and co-infected with FLAG-His 8-ETAR(21–406)/ETBR(27–424)-LgBiT, engineered Gαq/Gαi, Gβ1-15AA-HiBiT, Gγ2, and scFv16 baculoviruses at a 1:1:1:1:1 ratio. The cells were then harvested by centrifugation 48 h post-infection and stored at −80 °C for future use.
The frozen cells were thawed on ice and resuspended in lysis buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCl, 10% (v/v) glycerol, 10 mM MgCl2, 5 mM CaCl2, and supplemented with EDTA-free protease inhibitor cocktail (TargetMol). For ET-1-ETAR-Gq/ IRL1620-ETBR-Gi complex, cells were lysed by dounce homogenization and complex formation was initiated with the addition of 25 mU/mL Apyrase (Sigma-Aldrich) and 10 μM ET-1/ IRL1620 (GenScript) for 1.5 h at room temperature (RT). The membrane was then solubilized by adding 0.5% (w/v) lauryl maltose neopentyl glycol (LMNG, Anatrace) and 0.1% (w/v) cholesterol hemisuccinate (CHS, Anatrace) for 2 h at 4 °C. The sample was clarified by centrifugation at 80,000×g for 30 min and the supernatant was then incubated with anti-DYKDDDDK Affinity Beads (GenScript) for 3 h at 4 °C. After incubation, the resin was collected by centrifugation (600×g, 10 min) and loaded into a gravity flow column, followed by a wash with 20-column volumes of 20 mM HEPES, pH 7.5, 100 mM NaCl, 10% (v/v) glycerol, 5 mM MgCl2, 1 μM ET-1/IRL1620, 0.01% (w/v) LMNG, and 0.002% (w/v) CHS. The protein was finally eluted with ten-column volumes of 20 mM HEPES, pH 7.5, 100 mM NaCl, 10% (v/v) glycerol, 5 mM MgCl2, 1 μM ET-1/IRL1620, 0.01% (w/v) LMNG, and 0.2 mg/mL FLAG peptide. The purified complexes were concentrated using an Amicon Ultra centrifugal filter (molecular weight cut-off of 100 kDa, Millipore) and then subjected to a Superose 6 Increase 10/300 GL column (GE Healthcare) that was pre-equilibrated with buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 1 μM ET-1/IRL1620, 0.00075% (w/v) LMNG, 0.00025% (w/v) GDN, and 0.0002% (w/v) CHS. The purification procedure of ET-1-ETBR-Gq complex was similar to ETAR, expect the addition of 0.05% (w/v) digitonin, the final size-column equilibrated with 0.05% (w/v) digitonin instead of 0.00075% (w/v) LMNG, 0.00025% (w/v) GDN, and 0.0002% (w/v) CHS. The monomeric fractions of the complex were collected and concentrated for cryo-EM examination.
The frozen cells were thawed on ice and resuspended in lysis buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCl, 10% (v/v) glycerol, 10 mM MgCl2, 5 mM CaCl2, and supplemented with EDTA-free protease inhibitor cocktail (TargetMol). For ET-1-ETAR-Gq/ IRL1620-ETBR-Gi complex, cells were lysed by dounce homogenization and complex formation was initiated with the addition of 25 mU/mL Apyrase (Sigma-Aldrich) and 10 μM ET-1/ IRL1620 (GenScript) for 1.5 h at room temperature (RT). The membrane was then solubilized by adding 0.5% (w/v) lauryl maltose neopentyl glycol (LMNG, Anatrace) and 0.1% (w/v) cholesterol hemisuccinate (CHS, Anatrace) for 2 h at 4 °C. The sample was clarified by centrifugation at 80,000×g for 30 min and the supernatant was then incubated with anti-DYKDDDDK Affinity Beads (GenScript) for 3 h at 4 °C. After incubation, the resin was collected by centrifugation (600×g, 10 min) and loaded into a gravity flow column, followed by a wash with 20-column volumes of 20 mM HEPES, pH 7.5, 100 mM NaCl, 10% (v/v) glycerol, 5 mM MgCl2, 1 μM ET-1/IRL1620, 0.01% (w/v) LMNG, and 0.002% (w/v) CHS. The protein was finally eluted with ten-column volumes of 20 mM HEPES, pH 7.5, 100 mM NaCl, 10% (v/v) glycerol, 5 mM MgCl2, 1 μM ET-1/IRL1620, 0.01% (w/v) LMNG, and 0.2 mg/mL FLAG peptide. The purified complexes were concentrated using an Amicon Ultra centrifugal filter (molecular weight cut-off of 100 kDa, Millipore) and then subjected to a Superose 6 Increase 10/300 GL column (GE Healthcare) that was pre-equilibrated with buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 1 μM ET-1/IRL1620, 0.00075% (w/v) LMNG, 0.00025% (w/v) GDN, and 0.0002% (w/v) CHS. The purification procedure of ET-1-ETBR-Gq complex was similar to ETAR, expect the addition of 0.05% (w/v) digitonin, the final size-column equilibrated with 0.05% (w/v) digitonin instead of 0.00075% (w/v) LMNG, 0.00025% (w/v) GDN, and 0.0002% (w/v) CHS. The monomeric fractions of the complex were collected and concentrated for cryo-EM examination.
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