Expression and purification of CCL2–CCR2–Gi and apo CCR3–Gi complexes

In this work, a NanoBiT tethering strategy was employed for complex stabilization^25–27. For the purification of CCL2–CCR2–G_i, the sequence of human CCR2 (residues 1–355) was fused with a LgBiT subunit (Promega) at the C-terminus followed by a double MBP-tag via a GS linker containing a TEV protease cleavage site (ENLYFQG). The sequence of CCL2(1–72) was fused to the N-terminus of CCR2. For the expression of the apo CCR3–G_i complex, a constitutive active mutation was introduced (I244^6.40A). CCR3 was fused with a 6× His-tag at the N-terminus and a following BRIL (MBP) fusion protein via a linker containing a TEV protease site (ENLYFQG). And the sequence of prolactin precursor (PP) signal peptide was fused into the N-terminus of CCL3(4–69) and BRIL-CCR3. In this study, the human CCL11(1–74) was also purified for use in the formation of the CCR3–G_i complex. First, we cloned the cDNA sequence of CCL11(1–74) into a pETDuet vector with an N-terminal SUMO and 6× His-tag. The CCL11 expression vector was transformed into Escherichia coli BL21(DE3) cells. After overnight expression at 16 °C, an Ni-chelating HP Sepharose column (GE Healthcare) was used for purification of 6× His-SUMO-CCL11. Then, the purified protein was desalted and treated with ULP1 protease to remove the 6× His-SUMO-tag. The cleaved protein was passed through the Ni-chelating HP Sepharose column again, and CCL11 without the tag was collected as flow-through. Finally, CCL11 was purified by gel filtration chromatography with a HiLoad 26/60 Superdex 200 column (GE Healthcare) in 20 mM Tris, pH 8.0, 150 mM NaCl, and 10% glycerol.

The sf9 insect cells were infected with viruses encoding CCL2–CCR2–LgBiT/CCR3–LgBiT, Gα_i, Gβ1–peptide86, Gγ2, and scFv16 at a ratio of 1.5:1:1:1:2. After 48 h expression, the infected cells were collected and resuspended in 20 mM HEPES, pH 7.5, 100 mM NaCl, 20 mM KCl, 10 mM MgCl₂, and 5 mM CaCl₂ supplemented with an EDTA-free protease inhibitor cocktail (TargetMol). Then, 10 μM of purified CCL11 was added for the activation of CCR3, and the membrane was solubilized with 0.5% (w/v) n-dodecyl β-d-maltoside (DDM, Anatrace), 0.01% (w/v) LMNG, and 0.1% (w/v) CHS for 2 h at 4 °C. For the CCL2–CCR2–G_i complex, the supernatant was collected and incubated with amylose resin (NEB). After binding, the resin was packed into a gravity-flow column and washed in 20 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM MgCl₂, 0.01% (w/v) LMNG, 0.01% (w/v) GDN (Anatrace), and 0.004% (w/v) CHS. TEV protease was then added to remove the C-terminal MBP, the cleaved protein was passed through Amylose resin again, and the complexes without MBP were collected as flow-through. For the purification of the CCR3–G_i complex, the supernatant was isolated and incubated at 4 °C with TALON^® Metal Affinity Resin. After binding, the talon resin with protein complex was loaded onto a gravity-flow column. The talon resin was washed with 20 column volumes of 20 mM HEPES, pH 7.5, 100 mM NaCl, 25 mM imidazole, 0.01% (w/v) LMNG, 0.005% (w/v) GDN (Anatrace), and 0.004% (w/v) CHS and eluted with the same buffer plus 300 mM imidazole. The complex was concentrated and loaded onto a Superdex 200 10/300 GL Increase column (GE Healthcare), and monomeric CCL2–CCR2–G_i and apo CCR3–G_i complexes were collected in 20 mM HEPES, 100 mM NaCl, pH 7.5, 2 mM MgCl₂, 0.0005% (w/v) LMNG, 0.00025% GDN, and 0.0002% (w/v) CHS.