The futA gene sequence was subcloned into the pET21a vector (Novagen) and was transformed into E. coli BL21 (DE3) pLysS cells. Transformants were inoculated in LB medium supplemented with ampicillin (100 mg/ml) and grown at 37°C overnight. The cells were then transferred into fresh medium and grown at 37°C; when the OD600 reached 0.8 to 1.0, IPTG was added to a final concentration of 0.5 mM and induced expression overnight at 20°C. The cells were harvested and suspended in binding buffer [25 mM tris-HCl (pH 7.5), 100 mM NaCl, and 20 mM imidazole] and lysed by sonication on ice. The recombinant proteins were affinity-purified using a Ni-NTA (Ni2+-nitrilotriacetate) column (Smart-lifesciences, Changzhou, China) and eluted with buffer [200 mM imidazole, 500 mM NaCl, 100 mM tris-HCl (pH 8.5), and 10 mM 2-mercaptoethanol]. Proteins were further purified by gel filtration using a Superdex-75 column (GE Healthcare) equilibrated against buffer [25 mM tris-HCl (pH 8.0), 100 mM NaCl, and 1 mM dithiothreitol]. Last, the pure proteins were concentrated with a 30-kDa Vivaspin-20 concentrator (GE Healthcare) to ∼10 mg/ml.

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