Isopropyl β d 1 thiogalactopyranoside (iptg)

The ORF corresponding to RodZ (ORF wcw_0755) was amplified by PCR using the primers RodZ_fwd and RodZ_rev and cloned into the pET200 vector (Life technologies) in E. coli. This vector allows the expression of a N-terminal tagged version of the protein under a isopropyl-β-D-thiogalactopyranoside (IPTG) inducible promoter. Protein was expressed with 1 mM IPTG (MP Biomedicals, Santa Ana, CA) during 4 h and purified in presence of 8 M urea. Bacteria were lysed using Fastbreak Lysis buffer (Promega). Lysate was then incubated with MagneHis Ni particles beads and beads were washed with binding/wash buffer (Promega). The purified protein was then eluted using 500 mM imidazole (Sigma-Aldrich)56 (link). The purified protein was then used to immunize mice (Eurogentec, Seraing, Belgium).

LysDW2, THDW2, and truncated derivatives were overexpressed in the pQE-60/XL1 Blue expression system as per the Qiagen QIAexpressionist manual with minor changes. Twenty milliliters of superbroth containing 200 µg/ml was inoculated with 2% inoculum of overnight cultures of the DW2 peptidoglycan hydrolase clones and the empty pQE-60/XL1Blue which was used as a negative control. The cultures were incubated at 170 rpm at 37 °C until they reached an OD_590nm of 0.4–0.6. After cooling the samples on ice for 1 h, 1 mM of IPTG (MP Biomedicals, 114064112) was added. The cultures were then further incubated 37 °C for 5 h before being spun at 6000 g for 20 min and the supernatant was discarded. In a separate experiment clones were induced at 26 °C for 14 h. The cell pellets were washed with 25 mM Tris pH 8 and resuspended in bugbuster protein extraction reagent (Novagen, 70584-3) and shaken at 120 rpm for 20 min. The cells were then spun at 6000 g for 20 min and the soluble fraction was separated from the insoluble fraction which was subsequently resuspended in bugbuster.