Expression and Purification of Recombinant OxDC

Recombinant, His₆-tagged wt OxDC was expressed and purified following published procedures^{6 (link)} with minor modifications to avoid protein precipitation during concentration. Thus, expression was induced in the presence of 5 mM MnCl₂ after heat shocking the bacteria for 15 min at 42 °C with constant agitation. After the addition of IPTG, cultures were incubated at 37 °C for 5 h before being harvested by centrifugation at 2000g for 20 min (4 °C) and sonicated in lysis buffer. The resulting supernatant was centrifuged at 20000g for 20 min (4 °C) before the enzyme was purified from the cleared lysate by metal affinity chromatography on a Ni-NTA column. The eluted protein was subjected to dialysis at 4 °C to give a solution in 50 mM Tris buffer containing 20% glycerol and 500 mM NaCl (pH 8.5). Storage buffers also contained 20% glycerol. Protein concentrations were determined using the CoomassiePlus Protein Assay reagent obtained from ThermoFisher Scientific (Waltham, MA), and the metal content of purified, recombinant wt OxDC (Table S1) was obtained at the University of Georgia Center for Applied Isotope Studies Chemical Analysis Laboratory (Athens, GA).