Dioleoyl phosphatidylcholine and bovine heart cardiolipin were from Avanti Polar Lipids (Alabaster, AL). 2-Hydroxypropyl-β-cyclodextrin (HP-β-CD) was from Cerestar (Hammond, IN). Vitamin D3, NADPH and isocitric acid (tri-sodium salt) were from Sigma-Aldrich (Sydney, Australia) and 5β-cholestane-3α,7α,12α-triol was from Steraloids (Newport, RI). Lumisterol3 (L3) was purchased from Toronto Research Chemicals (Toronto, Canada) and was purified before use on a Brownlee Aquapore C18 column (25 cm × 1 cm, 10 μm particle size) using a gradient of methanol in water (64–100% methanol) for 15 min followed by 100% methanol for 45 min at a flow rate of 1.5 ml/min. (25R)-27-Hydroxycholesterol and (25S)-27-hydroxycholesterol were from Cayman Chemical (Ann Arbor, MI).
Human CYP27A1 with the N-terminal mitochondrial target sequence removed and a six histidine tag added to the C-terminus was expressed in E. coli and purified by Ni affinity- and octyl Sepharose-chromatography, as described previously [26 (link)]. Human adrenodoxin reductase, and mouse and human adrenodoxin were also expressed in E. coli and purified as described before [27 (link)–30 (link)].
Human CYP27A1 with the N-terminal mitochondrial target sequence removed and a six histidine tag added to the C-terminus was expressed in E. coli and purified by Ni affinity- and octyl Sepharose-chromatography, as described previously [26 (link)]. Human adrenodoxin reductase, and mouse and human adrenodoxin were also expressed in E. coli and purified as described before [27 (link)–30 (link)].