Expression in E. Coli, purification of recombinant membrane scaffold protein (MSP1D1), human hepatic CYP3A4 and rat NADPH-dependent CYP P450-reductase (CPR), and preparation of CYP3A4-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) nanodiscs (ND) were performed, as described previously [21 (link),22 ,42 (link),43 (link)]. CYP3A4 was available as a NF-14 gene construct in the pCWOri+ vector with a C-terminal penta-histidine affinity tag that was generously provided by Dr. F. P. Guengerich (Vanderbilt University, Nashville, TN, USA). Full length CPR was expressed while using a rat CPR/pOR262 plasmid, which was a generous gift from Dr. Todd D. Porter (University of Kentucky, Lexington, KY, USA). CYP3A4 was incorporated in ND from assembly mixture containing CYP3A4, MSP1D1 and POPC solubilized in cholate, as described [22 ,44 (link),45 (link)]. The detergents were removed by incubation with Amberlite XAD-2 (Millipore Sigma, Saint-Louis, MO, USA) for at least 4 h on ice. Further purification steps included nickel-affinity chromatography (Ni-NTA, Thermo Scientific, Schaumburg, IL, USA), followed by size-exclusion chromatography (Superdex 200HR10/30; GE Life Sciences, Chicago, IL, USA), as described before [22 ]. CPR was incorporated by direct addition to CYP3A4 ND at 4:1 molar access for functional studies, as described [46 (link)].
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