FMDV 3C Protease Purification Protocol

A pET28b plasmid encoding the Δ3B1-3B2-3B3-3C-His6 sequence from FMDV A₁₀61 with two mutations to enhance enzyme solubility (C95K/C142A) was obtained from Stephen Curry at Imperial College London (50 (link)). Escherichia coli BL21(DE3) pLysS was transformed with this plasmid and grown to an optical density at 600 nm of 0.4 to 0.7 absorbance units. Expression was induced by the addition of 1 mM isopropyl-β-d-thio-galactosidase (IPTG; Sigma) for 4 h at 37°C. Bacteria were harvested by centrifugation and resuspended in a lysis buffer containing 50 mM HEPES, pH 7.1, 200 mM NaCl, 1× HALT protease inhibitor cocktail (Thermo Fisher Scientific), 1% Igepal CA-630 (Sigma), 100 μg/ml lysozyme, 100 μg/ml DNase (Invitrogen) on ice for 1 h. Lysates were subjected to 12, 30-s sonication cycles at an amplitude of 10 μm (Microson XL-2000; Misonix) with 30 s on ice between each sonication. Lysates were clarified by centrifugation and His-tagged 3C^pro purified by nickel ion affinity chromatography using HisTrap columns (GE Healthcare). Elution fractions were analyzed by SDS-PAGE, and fractions with the highest concentration of purified protein were pooled and dialyzed against 50 mM HEPES, pH 7.1, 0.2 M NaCl, 1 mM EDTA, 1 mM β-mercaptoethanol, and 5% glycerol. Purified dialyzed proteins were stored at −80°C.