The lntBCG allele was deleted in the M. bovis BCG SmR chromosome as described previously [31, 32] and confirmed by Southern blot analysis with 0.2 kbp SalI lnt downstream probe. For complementation with M. bovis BCG BCG_2070c a 6.3 kbp fragment from M. bovis BCG from position 2289839 to 2296178 spanning the entire lnt gene was cloned into pGEM-T Easy (Promega) to result in pGEM-T Easy-lntBCG_2070c

and subsequently subcloned as a 6.3 kbp EcoRI fragment into the HpaI site of plasmid pMV361-hyg [33] to result in pMV361-hyg- lntBCG_2070c. Complementation was confirmed by Southern blot analyses with 0.2 kbp KpnI/HindIII lntBCG_2070c upstream probe. Expression of Lipoproteins LprF, LpqH, LpqL and LppX Plasmid pMV261-Gm, a derivative of pMV261 shuttle vector, is able to replicate selleck chemicals in E. coli as well as in mycobacteria [34]. LprF[13], lpqH, lpqL and lppX[12] were amplified by PCR from M. tuberculosis genomic DNA and fused to the M. tuberculosis 19 kDa promoter. The target proteins and 19 kDa promoter are identical between M. tuberculosis and M. bovis BCG. Sequences encoding a hemagglutinin and a hexa- histidine epitope were fused to the 3’ part of each gene to facilitate subsequent purification and detection on Western blot. The insert was cloned into the EcoRI site of pMV261-Gm to result in pMV261-Gm-LprF, pMV261-Gm-LpqH, pMV261-Gm-LpqL and pMV261-Gm-LppX. Subsequently

plasmids were transformed into BCG parental strain, Δlnt and Δlnt-lntBCG_2070c. Preparation AZD1480 clinical trial of cell Nutlin-3a molecular weight extracts and Western blot analysis Bacteria from 1-liter cultures were harvested and resuspended in phosphate-buffered saline containing Complete

EDTA-free tablets (Roche) to inhibit protein degradation. Cells were lysed by three French Press cycles (American Instrument Co.) at 1.1 x 106 Pa. Extracts were treated with 2% sodium N-lauroylsarcosine (SLS) for 1 h at room temperature, and incubated this website for 16 h at 4°C thereafter. Extracts corresponding to 1–5 μg of total protein were separated by a 12.5% SDS-PAGE gel and subsequently analyzed by Western blot using anti-HA-antibody (1:300, Roche) and corresponding secondary antibody conjugated with horseradish peroxidase. Fast protein liquid chromatography protein purification Soluble fractions of cell extracts from recombinant strains expressing epitope-tagged proteins were diluted with buffer containing 20 mM NaH2PO4, 0.5 M NaCl, pH 7.4 to 1% sodium N-lauroylsarcosine and loaded on a HisTrap™ HP column (GE Healthcare) previously equilibrated with buffer containing 20 mM NaH2PO4, 0.5 M NaCl, 0.2% sodium N-lauroylsarcosine and 20 mM imidazole, pH 7.4. Proteins were eluted applying an imidazole gradient (0.125-0.5 M). As a further purification step, if necessary, HisTrap™ HP column flow through was dialyzed against buffer containing 20 mM Tris-hydroxymethyl-aminomethane, 0.1 M NaCl, 0.1 mM EDTA, pH 7.5 and loaded onto anti-HA-affinity matrix (Roche). Proteins were eluted with buffer containing 0.