*4.3. Expression of ABCG5*/*G8 Missense Mutants in Pichia pastoris Yeast*

Both WT and mutant plasmids (20 mg each plasmid) were linearized using *Pme*I and co-transformed into the *Pichia* strain KM71H by electroporation. Immediately, the cells were resuspended with 1 mL of ice-cold 1 M sorbitol and incubated at 30 ◦C for 1 h. Then, 5 mL of fresh YPD was added and incubated for 6 h at 250 rpm and 30 ◦C. The cells were then centrifuged at 3000× *g* for 10 min and resuspended with 200 µL of YPD. Next, 100 µL of transformants were plated on YPDS plates containing 100 (low), 500 (medium), or 1000 (high) µg/mL Zeocin to screen for successful transformation. Seven colonies were picked and grown in 10 mL of MGY medium for 24 h in sterile 50 mL tubes at 250 rpm and 30 ◦C. The cells were centrifuged for 10 min at 3000× *g* and then resuspended with 10 mL of minimal methanol (MM) medium. Then, 50 µL of methanol was added to the medium and once again after 12 h. The cells were harvested after 24 h incubation at 250 rpm and 30 ◦C, resuspended in 600 µL mPIB buffer, and transferred into a 1.5 mL Eppendorf tube. After adding 500 µL of glass beads, protease inhibitors, and 10 mM DTT, the cells were lysed using a mini-bead beater (Biospec), with 1.5 min beating and 1.5 min rest on ice for three cycles. The unbroken cells and beads were pelleted by centrifugation at 5000× *g* for 5 min at 4 ◦C, followed by 21,130× *g* for 5 min at 4 ◦C. The supernatant was collected, and the concentration of the total proteins was estimated by Bradford assay. Next, 1 µL of cell lysate and 0–10 µg BSA standards were separately added to 200 µL of Bradford reagent on a 96-well plate. Absorbance at 595 nm was used to measure the protein concentrations using a Synergy H1 Hybrid reader (BioTek/Agilent, Santa Clara, CA, USA). The cell lysates (20 or 30 µg of total proteins) were resolved by SDS–PAGE, and protein expression was analyzed by immunoblotting using monoclonal anti-RGS-His antibodies (Qiagen Toronto, Toronto, ON, Canada) to detect ABCG5 and polyclonal anti-hABCG8 antibodies (Novus Biologicals, Centennial, CO, USA) to detect ABCG8. The clones expressing the highest level for both subunits were selected and stored in 20% glycerol at −75 ◦C.
