A C0-C7 construct with a C-terminal His-tag was codon-optimized for expression in Escherichia coli and was expressed using the pET-21 a (+) expression vector. Cells containing the C0-C7 plasmid were grown in Rosetta (DE3) pLysS cells, induced, and harvested as described by the manufacturer (Qiagen, Germany). The cells were then lysed in a lysis buffer containing 10 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, 1 mM β-mercaptoethanol, leupeptin (0.01 mg/ml), 1 mM phenylmethylsulfonyl fluoride, and Roche cOmplete protease inhibitor cocktail. Lysis was carried out on an emulsiflex or a sonicator, and the lysate was clarified by centrifugation at 35,000g for 35 min. The supernatant was loaded on a nickel–nitrilotriacetic acid (Ni-NTA) column (GE) on a fast protein liquid chromatography. The column was washed with 10 and 15 mM imidazole followed by a linear elution gradient of 15 to 400 mM imidazole. After analyzing the fractions on SDS–polyacrylamide gel electrophoresis (PAGE), pure fractions were pooled together and concentrated using an Amicon 10-kDa molecular weight cutoff filter. These concentrated fractions were then diluted into a buffer containing 10 mM imidazole (pH 7.5), 4 mM MgCl2, and 1 mM dithiothreitol (DTT) and loaded on an ion exchange Q-column (GE). The dilution step was essential to bring down the ionic strength to 60 to 70 mM for efficient attachment of the C0-C7 to the Q-column. The protein was eluted by a 0 to 100% linear NaCl gradient, and the peak fractions were analyzed by SDS-PAGE. Pure fractions were pooled, concentrated, and finally loaded on a preparatory grade Superdex S200 (16/600) size exclusion column. The C0-C7 eluted as a peak from the size exclusion chromatography column with minor shoulder peaks. Only the major peak fractions were collected, analyzed by SDS-PAGE, pooled, and subsequently used for MST experiments. The protein was eluted in a buffer containing 10 mM imidazole (pH 7.5), 100 mM potassium acetate, 4 mM MgCl2, 1 mM EDTA, and 1 mM DTT.

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