Lipidex 5000

The crude extract in the methanol:dichloroethane:H₂O 85:10:5 (by vol) phase was dried under reduced pressure at 37°C and then resuspended in a 8–10 ml, corresponding to 20–25% of the column volume, of the same solvent mixture. If the solution was not clear the sample was centrifuged at 200 x g for 5 min to remove non-dissolved NaCl and the supernatant was loaded on a 2.5 cm x 8 cm Lipidex-5000 (PerkinElmer, Waltham, MA, USA) column equilibrated in methanol:dichloroethane:H₂O 85:10:5 (by vol). Fractions were collected, dried under reduced pressure at 37°C, and weighed. The first fraction corresponding to 0.7–1 column volume, containing rSP-C33Leu as well as polar lipids, was dried, redissolved in about 4 ml of methanol:dichloroethane:H₂O 85:10:5 (by vol), corresponding to 1% of the column volume and centrifuged at 70 x g for 5 min to further remove NaCl. The supernatant was loaded on a 2.5 cm x 80 cm Lipidex-5000 column in methanol:dichloroethane:H₂O 85:10:5 (by vol). One fraction of 100 ml followed by fractions of 10 ml were collected, dried under reduced pressure and weighed. An aliquot of each fraction was analyzed by SDS-PAGE as described above. Fractions that contained rSP-C33Leu were dried and stored at -20°C.

Kanamycin, sodium chloride, potassium chloride, Tris, Hepes, potassium phosphate, glycerol, benzonase, lysozyme, thrombin, protease inhibitor tablets, Coomasssie Brilliant Blue and atRA were purchased from Millipore-Sigma (St. Louis, MO, USA). The 4-oxo-atRA-d₃ was purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and 4-OH-atRA was purchased from Toronto Research Chemicals (North York, ON, Canada). Sodium hydroxide, PMSF, imidazole, Pierce BCA protein assay, DTT, EDTA, IPTG, tryptone, yeast extract, high-performance liquid chromatography (HPLC) and mass spectrometry grade acetonitrile, water, and ethyl acetate were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Lipidex-5000 was purchased from Perkin Elmer Inc (Waltham, MA, USA). Mini-PROTEAN TGX protein gels were purchased from Bio-Rad (Hercules, CA, USA). NBD-stearate was purchased from Avanti Polar Lipids (Birmingham, AL, USA). Pooled human liver microsomes were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Recombinant CYP3A4 and CYP2C8 Supersomes co-expressed with cytochrome P450 reductase and cytochrome b5 were purchased from Corning (Glendale, AZ, USA). Recombinant CYP26A1 was expressed in Sf9 insect cells, as previously described [17 (link),20 (link)]. Rat P450 reductase was expressed in E. coli and purified, as previously described [33 (link)].