The N-terminal (His)X6 and maltose-binding protein (hisMBP)–tagged wild-type mmoD (MMOD) was prepared in E. coli Rosetta (DE3) with auto-inducible media (33). Cell pastes were resuspended in solution containing tris-HCl (pH 7.5, 30 mM), NaCl (500 mM), and β-mercaptoethanol (5 mM) with protease inhibitor cocktails. After sonication on ice for 3 min, soluble lysate was recovered by centrifugation at 20,000g for 30 min and was subsequently applied to a cobalt affinity resin (Takeda). The hisMBP-MMOD proteins were eluted with elution buffer [tris-HCl, 30 mM (pH 7.5); NaCl, 500 mM; imidazole, 300 mM; and β-mercaptoethanol, 5 mM]. To remove the hisMBP tag, we mixed the elution fraction and tobacco etch virus protease (1:100 ratio) together in the amylose resin. After overnight incubation at 4°C, the flow-through fraction, which contains MMOD, was collected from the amylose resin and loaded onto an HP Q column (GE Healthcare); equilibrated with Hepes (pH 7.5, 30 mM), NaCl (100 mM), and DTT (1 mM); and eluted in a 0 to 1000 mM NaCl gradient. On the basis of the results of SDS–polyacrylamide gel electrophoresis, fractions containing MMOD were pooled. The pooled fractions were then concentrated using Amicon (EMD Millipore) and applied to a Superdex 200 (10/300) size exclusion chromatography (GE Healthcare) column equilibrated in Hepes (pH 7.5, 30 mM), NaCl (100 mM), and tris(2-carboxyethyl)phosphine (TCEP, 1 mM).

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