Overexpression and Purification of Bacillus subtilis P5C Reductases

The sequences coding for the four putative P5C reductase proteins from Bacillus subtilis strain 168 were amplified by PCR using the primers reported in Table 1, and cloned into vector pMCSG68 according to the standard protocol described previously (Eschenfeldt et al., 2013 (link); Forlani et al., 2015b (link)). The pMCSG68 vector introduces a His₆-tag followed by the Tobacco Etch Virus (TEV) protease cleavage site at the N-terminus of the expressed protein. The correctness of the insert was confirmed by DNA sequencing. Overexpression was carried out in BL21 Gold E. coli cells (Agilent Technologies). The bacteria were cultured with shaking at 210 rpm in LB medium supplemented with 150 μg mL⁻¹ ampicillin at 37°C until the OD₆₀₀ reached 1.0. The temperature was lowered to 18°C, and isopropyl-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM. The culture was grown for 18 h and then centrifuged at 4,000 g for 10 min at 4°C. The cell pellet from 1 L culture was resuspended in 35 mL of lysis buffer (50 mM HEPES sodium salt pH 8.0, 500 mM NaCl, 5% glycerol, 20 mM imidazole, 10 mM β-mercaptoethanol) and stored at −80°C.