Cloning and Expression of MotA Protein from Aquifex aeolicus

The full-length motA gene from A. aeolicus (motA^Aa) was cloned into a pColdI vector (Takara Bio Inc., Kusatsu, Japan) as previously described24 (link). MotA^Aa protein was expressed with an N-terminal histidine tag in E. coli BL21-CodonPlus(DE3)-RIPL (Agilent Technologies, Santa Clara, CA, USA) cells. The cells were grown in 1.5 L of SB medium [1.2% (w/v) Bacto tryptone, 2.4% (w/v) Bacto yeast extract, 0.5% (w/v) glycerol, 1.25% (w/v) K₂HPO₄, 0.38% (w/v) KH₂PO₄] containing 100 μg/mL ampicillin, at 37 °C, to an OD₆₆₀ of 0.6–0.8, and 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) was subsequently added to the culture after cooling on ice for 30 min before the culture was prolonged for about 20 h at 16 °C. Cells were collected by centrifugation (7,000 × g) and then resuspended in 50 mL of TN buffer [50 mM Tris-HCl (pH 8.0], 200 mM NaCl) containing half a protease inhibitor cocktail tablet (Roche diagnostics) and about 10 mg of lysozyme (Wako, Osaka, Japan). Cells were then disrupted by sonication and ultracentrifuged at 100,000 × g for 30 min, and the pellet (membrane fraction) resuspended in TN buffer.