Purification of CauErg11 and CauMdr1 Proteins

Cells were harvested from 2 L YPD cultures, crude membranes prepared, and CauErg11-6×His, CauErg11 Y132F-6×His, CauErg11 K143R-6×His, or CauMdr1-6×His partially purified by nickel–nitrilotriacetic acid (Ni-NTA) affinity chromatography, essentially as described by Monk et al. [35 (link)]. The crude membranes were extracted in pH 7.5 solubilization buffer (10% (w/v) glycerol, 250 mM NaCl, 20 mM Tris, 0.5 mM PMSF, 1 tablet cOmplete^TM Mini, EDTA-free Protease Inhibitor cocktail (Roche, Basel, Switzerland) per 50 mL plus 16 mM n-decyl-β-D-maltopyranoside (DM Anagrade, Anatrace Products, LLC., Maumee, OH, USA). The detergent-solubilized protein affinity purified by binding to 2 mL of Ni-NTA Agarose matrix (Qiagen, Hilden, Germany) per g of protein in a disposable Econo-Pac^® Chromatography column (Bio-Rad) was washed with 2 mM L-histidine and eluted with 100 mM L-histidine. The eluted protein was concentrated to 500 µL using Amicon^® Ultra–4 Centrifugal Filter Units with a 50 kDa cutoff (Merck Millipore Ltd., Tullagreen, Ireland). Samples (1 µL) were separated by SDS-PAGE to obtain Coomassie-blue-stained protein bands for mass spectrometry of tryptic fragments. Tryptic fragments from the SDS-PAGE separated CauCdr1-6×His-containing band in crude membranes were used for mass spectrometry. For protein identification, the obtained fragments were searched against the Swiss-Prot database, the sequence of interest, and S. cerevisiae database to exclude possible background contamination.