Bl21 gold e coli cells

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.

Full-length PARIS WT (aa 1-644) and XIAP were cloned into the pGEX-6p-1 vector that was then used to transform BL21 gold E. coli cells (Agilent Technologies, Santa Clara, CA, USA). The transformed cells were grown at 37 °C until the optical density reached 0.6, followed by cooling on ice for 1 h. The transformed E.coli cells were further grown in the presence of 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) for 3 h at 37 °C. Cells were then harvested by centrifugation at 10,000× g for 5 min at 4 °C and lysed by sonication in the lysis buffer (4 M NaCl, 10% NP40, 1 M Tris-HCl pH 7.4, 0.5 M EDTA, 0.25 M EGTA, 0.1% β-mercaptoethanol, and 100 mM PMSF). The protein samples were centrifuged at 10,000 × g for 15 min, mixed with glutathione sepharose 4B (Sigma-Aldrich, St. Louis, MO, USA), and incubated at 4 °C overnight. The beads were then washed using the wash buffer (4 M NaCl, 10% NP40, 1 M Tris-HCl pH 7.4, 0.5 M EDTA, and 0.25 M EGTA) on ice, and the bound proteins were eluted with the elution buffer (40 mM glutathione, 1 M HEPES, 1 M NaCl, 100% glycerol, and 10% Triton X-100) for 1 h at 4 °C. The purity of GST-PARIS and GST-XIAP recombinant proteins were analyzed by SDS-PAGE, and concentration was determined using the Bradford assay.