Purification of Recombinant Voltage-gated Ca2+ Channels

The pFastBac-Flag-Ca_VAb was used as the construct for producing homotetrameric model voltage-gated Ca²⁺ channel^{19 (link)}. I199S, W195Y, and T206S constructs were generated via site-directed mutagenesis using QuickChange (Stratagene). Recombinant baculovirus were produced using the Bac-to-Bac system (Invitrogen), and T. ni insect cells were infected for large-scale protein purification. Cells were harvested 72 h post-infection and re-suspended in buffer A (50 mM Tris-HCl, pH = 8.0, 200 mM NaCl) supplemented with protease inhibitors and DNase. After sonication, digitonin (EMD Biosciences) was added to 1%, and solubilization was carried out for 1–2 h at 4 °C. Clarified supernatant was then incubated with anti-Flag M2-agarose resin (Sigma) for 1–2 h at 4 °C with gentle mixing. Flag-resin was washed with ten column volumes of buffer B (buffer A supplemented with 0.12% digitonin) and eluted with buffer B supplemented with 0.1 mg ml⁻¹ Flag peptide. The eluant was concentrated and then passed over a Superdex 200 column (GE Healthcare) in 10 mM Tris-HCl pH =8.0, 100 mM NaCl and 0.12% digitonin. The peak fractions were concentrated using a Vivaspin 30K centrifugal device.