Superdex 200 hr 10 30

5 μM of the R.PabI dimers and 2.5 μM of the nonspecific dsDNA (5′-GCACTAGTTCGAACTAGTGC-3′) were mixed in 10 mM MES (pH 6.0) and 300 mM NaCl. 5 μM of the R.PabI dimers (Y68F, R32A E63A, and Y68F R70D), 2.5 μM of the nonspecific dsDNA, and the R.PabI-nonspecific dsDNA mixtures were loaded onto a Superdex 200 HR 10/30 (GE Healthcare) column and were eluted with buffer containing 10 mM MES (pH 6.0) and 300 mM NaCl. 5 μM of the R.PabI dimers (Y68F D71R and Y68F R70D D71R) were loaded onto a Superdex 200 HR 10/30 (GE Healthcare) column and were eluted with buffer containing 10 mM MES (pH 6.0) and 600 mM NaCl to avoid nonspecific binding to the Superdex 200 column. To estimate the oligomeric state of R.PabI, the following standard proteins were used: ferritin (Mr = 440,000), aldolase (Mr = 158,000), conalbumin (Mr = 75,000), ovalbumin (Mr = 44,000), and ribonuclease A (Mr = 13,700).

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)].

All chemicals, Superdex 200 HR 10/30 (17-5175-01), Protein A-Sepharose (17046901), Protein G-Sepharose (17061801), and columns were obtained from GE Healthcare Life Sciences (New York, NY, USA). These preparations were free from possible contaminants. The MOG_35–55 (2568/1) was obtained from EZBiolab (Munich, Germany), while the Bordetella pertussis toxin (M. tuberculosis; BML-g100-0050) was obtained from the Native Antigen Company (Oxfordshire, UK).