Lauryl maltose neopentylglycol lmng

We used a similar expression and purification process as described before.^{24 (link)} Briefly, baculovirus encoding GPR3–LgBiT–TEV–2× MBP, Gα_s, Gβ₁, and Gγ₂ proteins were co-infected into Sf9 cells. Two days later, cells were collected and resuspended in a lysis buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl₂, 20 mM KCl and 5 mM CaCl₂. The mixture was incubated for 90 min at RT with apyrase (0.5 mU/mL) and Nb35 (20 µg/mL). Then, membrane was solubilized in buffer of 0.5% (w/v) lauryl maltose neopentylglycol (LMNG; Anatrace) and 0.1% (w/v) CHS-Tris for 2 h at 4 °C, followed by ultracentrifugation at 45,000 rpm at 4 °C for 45 min twice. The supernatant was loaded on an amylose column for 2 h, flowed out, washed with buffer solution containing 25 mM HEPES, pH 7.5, 150 mM NaCl and 0.01% LMNG/0.002% CHS, and eluted by the same wash buffer added with 10 mM maltose. After concentration and TEV cleavage overnight at 4 °C, the complex protein was loaded onto a Superdex 200 Increase 10/300 GL (GE Health Sciences) gel infiltration column pre-equilibrated with the buffer of 25 mM HEPES, pH 7.5, 150 mM NaCl, 0.00075% (w/v) LMNG, 0.00025% glyco-diosgenin and 0.0002% CHS (w/v) (Anatrace). The complex fractions were concentrated to 10 mg/mL and snap-frozen for grid preparation.

Most chemicals and molecular biology reagents were purchased from Sigma-Aldrich unless mentioned otherwise. HEK-293 cells (ATCC; cat. no. CRL-3216) were maintained at 37 °C under 5% CO₂ in Dulbecco’s modified Eagle’s medium (Gibco; cat. no. 12800-017) supplemented with 10% FBS (Gibco; cat. no. 10270-106) and 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin (Gibco; cat. no. 15140-122). Cells were cultured in 10 cm dishes (Corning; cat. no. 430167) at 37 °C under 5% CO₂ and passaged at 70 to 80% confluency using 0.05% trypsin-EDTA for detachment. Sf9 cells (Expression Systems; cat. no. 94-001 F) were maintained as suspension cultures in ESF 921 media (Expression Systems; cat. no. 96-001-01). Lauryl Maltose Neopentyl Glycol (LMNG) was purchased from Anatrace (cat. no. NG310).