hTRPV6 and rTRPV6 were expressed and purified as previously described for TRPV6 (13, 17, 28). In brief, hTRPV6 and rTRPV6 in pEG BacMam plasmids were used to generate baculovirus in Sf9 insect cells. This virus was used to transduce human embryonic kidney (HEK) 293 GnTI cells grown in suspension. Forty-eight to 72 hours after transduction, HEK cells were harvested and washed with phosphate-buffered saline (pH 8.0). Cells were broken by sonication, and cellular membranes were pelleted using ultracentrifugation. rTRPV6 was extracted from cellular membranes using 10 mM lauryl maltose-neopentyl glycol (LMNG) and purified using Strep-Tactin affinity chromatography, followed by size exclusion chromatography in 0.5 mM LMNG. hTRPV6 was extracted from cellular membranes using 20 mM n-dodecyl-β-d-maltopyranoside (DDM) and 0.2 mM cholesteryl hemisuccinate (CHS) supplemented with 20 μM purified CaM and 5 mM Ca2+ for ~2 hours. To maintain the integrity of the hTRPV6-CaM complex, 20 μM CaM was present throughout the entire purification, including affinity and size exclusion chromatography and subsequent amphipol trapping steps.

CaM was subcloned into the pCDEF-duet expression vector that was used to transform BL21(DE3). CaM was purified in two steps, using talon affinity and size exclusion chromatography. The 2L culture of CaM-expressing BL21(DE3) cells was grown in the presence of streptomycin (30 μg/ml) at 37°C for ~2.5 to 3.0 hours. Subsequently, CaM expression was induced by the addition of 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) at 20°C, and cells were grown for another 12 to 14 hours at 200 rpm and 20°C. Cells were harvested by centrifugation, resuspended in 40 ml of lysis buffer [20 mM tris-Cl (pH 8.0) and 150 mM NaCl], and subjected to sonication. The soluble fraction of the resulting slurry containing CaM was recovered by centrifugation. The N-terminally His-tagged CaM was first purified using affinity chromatography. The clarified CaM supernatant was loaded onto a 5-ml TALON metal affinity resin (Clontech) preequilibrated in lysis buffer. After overnight incubation at 20°C, the resin was washed with 30 ml of lysis buffer containing 20 mM imidazole and eluted with lysis buffer containing 250 mM imidazole. The N-terminal His-tag was removed by PreScission Protease at a 1:100 (CaM/PreScission Protease) ratio and again passed through the TALON column to remove the PreScission Protease and the cleaved tag from the CaM-containing solution. Subsequently, concentrated protein was loaded on the Superdex 200 gel filtration column (GE Healthcare) equilibrated with 1.5 column volume of the lysis buffer. CaM fractions were collected and concentrated for structural studies.

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