Recombinant CYP2C9 Protein Production

We cultured 293FT cells (Thermo Fisher Scientific) in Dulbecco’s modified Eagle’s medium (Nacalai Tesque) containing 10% fetal bovine serum at 37 °C under 5% CO₂. Cells were plated at a density of 2.2 × 10⁶ cells/100-mm dish; 24 h after plating, cells were transfected with plasmids encoding CYP2C9, CPR, and cytochrome b₅ cDNA (9.6 μg, 0.2 μg, and 0.2 μg, respectively) using 30 μL of 1.0 mg/mL Polyethylenimine “Max” (Polysciences, Inc., Warrington, PA, USA), according to previously described methods [32 (link)]. After incubation for 12 h at 37 °C, 0.25 mM 5-aminolevulinic acid hydrochloride (Nacalai Tesque) and 0.25 mM iron (II) sulfate heptahydrate (FUJIFILM Wako Pure Chemical Corporation) were added to the medium. After incubation for 48 h post-transfection at 37 °C, the cells were scraped from the plates. Microsomal fractions were prepared, and protein concentrations were determined as described previously [32 (link)].