The resultant two PCR products were used as templates for an overlapping extension PCR involving primers AA357 and AA354. The final PCR amplicon was then digested with both BamHI and SacI and ligated into pWW115 [52] that had been digested with these same restriction enzymes. The ligation mixture was used to transform O12E.mcbC::kan. A plasmid isolated from a spectinomycin-resistant colony and which expressed PARP inhibitor the His-tagged McbC protein was designated pAA111. Plasmid pWW115 was used to transform M. catarrhalis O12E.mcbC::kan to provide a negative control. Purification and detection of the His-tagged McbC protein M. catarrhalis

O12E.mcbC::kan(pWW115) and M.

catarrhalis O12E.mcbC::kan(pAA111) were grown independently in 1 L BHI overnight at 37°C with shaking. The cultures were subjected to centrifugation to pellet the bacterial cells and the supernatant fluid was filter-sterilized. Two columns each containing 1.5 mL of NiNTA agarose beads (Qiagen, Valencia, CA) were washed with washing buffer (50 mM NaH2PO4, 200 mM NaCl, 5 mM imidazole [pH 7.9]). The culture supernatant fluids were passed through the columns twice after which the columns were washed with washing buffer again. The His-tagged protein was eluted using elution buffer (50 mM NaH2PO4, 200 mM NaCl, 200 mM imidazole [pH 7.9]). not Selected fractions were pooled and dialyzed against PBS. SDS-digestion DMXAA datasheet buffer was added to a final concentration of 1× to each sample. For Western blot analysis, proteins were resolved by SDS-PAGE using 15% (wt/vol) polyacrylamide separating gels and transferred to polyvinylidene

difluoride membranes. The anti-His tag antibody HIS.H8 (Millipore, Temecula, CA) was used at a dilution of 1:2,000 in PBS-Tween containing 3% (wt/vol) dried milk and incubated with the membrane for 2 h at room temperature. Horseradish peroxidase-conjugated goat anti-mouse antibody (Jackson Immunoresearch, West Grove, PA) was used as the secondary antibody. The antigen-antibody complexes were detected by using Western Lightning Chemiluminescence Reagent Plus (New England Nuclear, Boston, MA). Construction of a plasmid containing the mcbI gene Primers AA353 (5′-ATGGATCCGAAAACTCATTGGGGAGATAGAGGGAT-3′) (BamHI site underlined) and AA378 (5′-TTGTGAGCTCGCTCGGATTTGCTATTATTGA-3′) (SacI site underlined) were used to PCR-amplify a 288-bp fragment containing the mcbI gene from M. catarrhalis O12E chromosomal DNA. The resultant PCR product was digested with both BamHI and SacI and ligated into pWW115 which had been digested with the same two restriction enzymes.