The E. coli strain T7 pLysS (New England Biolabs) was used as the host for heterologous expression of all MD variants. E. coli cells were transformed with pDEST14 derivatives encoding P MD variants with a C-terminal hexahistidine tag. Transformed cells were grown overnight at 37°C in Luria-Bertani broth medium supplemented with ampicillin (100 μg/ml) and chloramphenicol (34 μg/ml) and then diluted 1:40 in 1 liter of Super Broth medium supplemented with ampicillin and chloramphenicol. Cultures were incubated at 37°C until the OD600 (optical density at 600 nm) reached 0.7, and then protein expression was induced by the addition of 0.5 mM isopropyl-β-d-thiogalactopyranoside. After induction, cultures were incubated for 4 hours at 37°C. Bacteria were harvested by centrifugation at 4000g for 20 min and then resuspended in 30 ml per liter of culture of buffer A [50 mM tris-HCl (pH 8), 1 M NaCl, and 10 mM imidazole] supplemented with lysozyme (0.1 mg/ml), deoxyribonuclease (10 μg/ml), and 20 mM MgSO4 and stored at −20°C. The cells were disrupted by sonication, and the lysate was clarified by centrifugation at 12,000g for 45 min at 4°C. The soluble fraction was loaded onto 2 ml of Sepharose Fast Flow resin preloaded with nickel and pre-equilibrated with buffer A. The resin was then washed successively with 20 column volumes of buffer A and then with 20 column volumes of buffer A containing 20 mM imidazole. Proteins were eluted with 5 column volumes of buffer B [50 mM tris-HCl (pH 8), 1 M NaCl, and 500 mM imidazole]. Proteins that were soluble at pH 8 were directly concentrated on a 10-kDa concentrator (Ultracel, Amicon), then loaded onto a S75 16:60 SEC column, and eluted in 10 mM tris-HCl (pH 8) and 150 mM NaCl. Proteins that were soluble at acidic pH were dialyzed against 50 mM sodium acetate (pH 4.5) and 150 mM NaCl and then concentrated and purified by SEC using this buffer. The fractions were analyzed by 18% SDS-PAGE, and those containing the protein of interest were pooled, concentrated as described above, and stored at 4°C.

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