Decyl β d maltopyranoside dm

For overexpression and biochemical assays, the coding sequences of CLC^F ORF 1289 or ORF 1290 were separately cloned into a pASK vector with a C-terminal hexahistidine tag (22 (link)) and transformed into Escherichia coli (BL21-DE3). Protein expression was induced at OD₆₀₀ 0.5 with 0.5 mg/mL anhydrotetracycline for 3 h. After cell disruption by sonication, the protein was extracted with 2% (wt/vol) decyl-β-D-maltopyranoside (DM; Anatrace, Maumee, OH) for 2 h at room temperature. The lysate was centrifuged to pellet cell debris, and the protein was purified using cobalt affinity resin (1 mL/L culture, Takara Bio USA, San Jose, CA). The protein was washed with 100 mM NaCl, 20 mM imidazole, 20 mM Tris-HCl pH 8.0, 5 mM DM, and eluted in the same buffer with 400 mM imidazole, before a final size exclusion purification step (Superdex 200) in 100 mM NaCl, 10 mM NaF, 20 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) pH 7.5, 4 mM DM. To prepare proteoliposomes, 25 µg purified protein was mixed with 5 mg of E. coli polar lipid extract (Avanti Polar Lipids, Alabaster, AL) solubilized in 35 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and dialyzed against 0.3 M KF, 15 mM HEPES pH 7.0 at room temperature for 36 h with three buffer changes. Liposomes were stored in aliquots at −80°C until the day of use.