Nickel nitrilotriacetic acid ni nta resin

Cysteine-less Glt_Ph (CLGlt_Ph) and double cysteine mutants were transformed into Escherichia coli (E. coli) Top10 cells (Invitrogen). Bacterial cells were inoculated in Luria Broth medium containing 100 μg/mL ampicillin at 37 °C until OD₆₀₀ ~0.6–0.8. Protein expression was induced with 0.1% L-arabinose and harvested 4 h post-induction. All protein purification experiments were conducted at 4 °C. Membranes were first isolated and solubilized with 10 mM n-Dodecyl-β-D-maltoside (C₁₂M; Anatrace) in binding buffer containing 20 mM HEPES-Tris (pH 7.5), 200 mM NaCl, and 5 mM Na-Asp for 1 h. Proteins were purified using nickel-nitrilotriacetic acid (Ni-NTA) resin (Qiagen) and the histidine tag was subsequently removed by thrombin digestion (10 U/mg of protein). The proteins were further purified by size exclusion chromatography (SEC) using Superdex200 16/60 HiLoad gel filtration column (GE Healthcare). For CLGlt_Ph, Glt_Ph-XL1, the column was equilibrated with SEC running buffer containing 20 mM HEPES-Tris (pH 7.5), 25 mM NaCl, 25 mM KCl, 5 mM Na-Asp and 7 mM n-decyl-β-D-maltopyranoside (C₁₀M; Anatrace). For Glt_Ph-XL2 and Glt_Ph-XL3, the SEC buffer contained 10 mM HEPES-KOH/NaOH (pH 7.5), 100 mM NaCl, 0.1 mM Na-Asp and 7 mM C₁₀M. Proteins were concentrated to 6–7 mg/mL for gel analysis and crystallization.