Recombinant Expression and Purification of EhV-ATPase

Expression and purification of recombinant EhV-ATPase were performed as previously described^{41 (link)}. In brief, E. hirae V-ATPase was expressed recombinantly in Escherichia coli and purified by affinity purification with a Ni⁺-NTA (nitrilotriacetic acid) column (Ni⁺-NTA Superflow; Qiagen, Hilden, Germany). The column was preconditioned with buffer consisting of 50 mM potassium phosphate, 100 mM KCl, 5 mM MgCl₂, 20 mM imidazole, 10% glycerol, 0.05% n-dodecyl-β-D-maltoside (βDDM) at pH 7.5. The column was washed, and protein was eluted with buffer containing 50 mM potassium phosphate, 100 mM KCl, 5 mM MgCl₂, 300 mM imidazole, 10% glycerol, 0.05% βDDM at pH 7.5. The purified protein was concentrated using an Amicon Ultra 100 K filter (Merck Millipore, Billerica, Massachusetts, USA) before further purification using Superdex 200 gel filtration (GE Healthcare, Little Chalfont, UK) preconditioned with 50 mM Tris-HCl, 5 mM MgCl₂, 10% glycerol, 0.05% βDDM at pH 7.5. The rotor-rotating activity of the EhV-ATPase in presence of ATP, Na⁺, Mg²⁺ was confirmed by inhibition of ATP hydrolysis of the V₁ domain by DCCD, an inhibitor of c-ring rotation in the V_o domain^{33 (link)}.