*4.5. Purification of ABCG5*/*G8 and Its Mutants*

Both WT and mutants were purified following a protocol described previously [21], with minor modification. Briefly, DDM-solubilized membranes were subjected to a tandem affinity column chromatography, first using Ni–NTA and then CBP. The *N*-linked glycans and the CBP tag remained on the purified heterodimers, and the CBP eluates were further purified by gel-filtration chromatography using a Superdex 200 Increase 10/300 GL column (Cytiva) on an KTA Pure purification system (Cytiva, formerly GE Healthcare Life Sciences). The proteins in the peak fractions were collected and concentrated to 1–3 mg/mL for storage at −75 ◦C. Noticeably, the final yield for mutants was lower than WT, in a range of 400–800 µg per 6 L of cells. The expression level of the mutant proteins in the microsomes and their solubility were slightly lower than for WT. Some proteins were also lost during Ni–NTA binding and imidazole wash. The profile of the gel-filtration chromatography often showed a higher peak at the void volume than dimeric proteins. These factors collectively suggest that the mutant proteins were more prone to aggregation, thus explaining the lower yields.
