Expression and purification of proteins

All recombinant proteins expressed in E. coli or C. crenatum SPYA were labeled with a His-Tag at C-terminally. In order to express arginine decarboxylase, recombinant E. coli were cultured at 37 °C in LB medium until OD₆₀₀ reached about 0.6. Then, isopropyl β-d-1-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mM, and the strains were cultured at 16 °C for 12 h to induce the expression of the protein. The C. crenatum cells were cultured and induced in LBG medium for 18 h at 30 °C. After cultivation, the cells were collected by centrifugation at 8000×g and 4 °C for 5 min, washed twice and suspended in Tris-HCl buffer (pH 8.0). The cell suspension was disrupted on ice by sonication and the cell crude extract was obtained by centrifugation at 10,000×g and 4 °C for 20 min. After that, the proteins were purified using the HisTrap^TM HP affinity column (5 mL) and the AKTA protein purifier (GE Healthcare, Sweden). The target proteins were eluted by buffer M₀ (20 mM Tris-HCl, 500 mM NaCl, pH 7.4) and M₇₀₀ (20 mM Tris-HCl, 700 mM imidazole, 500 mM NaCl, pH 7.4) with a gradient ratio. The purified enzymes were analyzed by SDS-PAGE.