*4.4. Cell Culture and Microsomal Membrane Preparation*

The conditions for cell growth and WT protein induction were as previously described [21]. Briefly, cells were initially grown at 30 ◦C to accumulate cell mass in an Innova R43 shaker (Eppendorf) at 250 rpm for 24–48 h with the pH maintained at pH 5–6. To induce protein expression, cells were left fasting for 6–12 h, and then incubated with 0.1% (*v*/*v*) methanol for 6–12 h at 20 or 28 ◦C. The methanol concentration was increased to 0.5% (*v*/*v*) by adding methanol every 12 h for 48–60 h. Cell pellets were collected and resuspended in mPIB and stored at −75 ◦C. Approximately 45 ± 10 g of cell mass was typically obtained from 1 L of cultured cells. The frozen cells were thawed and lysed using a C3-Emulsifier (Avestin, Ottawa, ON, Canada) in mPIB in the presence of 10 mM DTT and protease inhibitors (1 µg/mL leupeptin, 1 µg/mL pepstatin A, 1 µg/mL aprotinin, and 2 mM PMSF(phenylmethylsulfonyl fluoride). The microsomal membranes were then prepared as previously described [21].
