Methods Colicin M expression and isolation Prior to isolation of colicin M, the cma colicin M structural and cmi immunity genes were
PCR amplified from the natural colicin M coding plasmid pCHAP1 using the primers ColM1 5′-TCACTCGAGCATGGAAACCTTAACTGTTCATGCA-3′ and ColM2 5′-CCACGCGTCCACTTCACAGTATGCTCACATTG-3′. The amplified fragment was digested selleck chemicals with the XhoI and MluI restriction enzymes and cloned into the pET8c expression vector, also cut with the same two enzymes . The isolated plasmid was designated pColM-imm Cloning of cma into the pET8c vector introduced an N-terminal histidine tag with expression under the control of a T7 promotor. Colicin M and the immunity protein were subsequently expressed in E. coli BL21 (DE3)pLysS and colicin M was purified using nickel affinity chromatography [80, 81]. For large scale
isolation an overnight culture of BL21 (DE3)pLysS, with plasmid pColM-imm was diluted 100 fold in 500 ml LB with ampicillin (120 μg/ml) and grown at 37°C to an OD600 0.6-0.8, when chromosomal T7 polymerase production was induced by addition of 0.8 mM IPTG and incubated for a further 4 h. Subsequently, cells were harvested this website and resuspended in 50 mM phosphate, 300 mM NaCl buffer, pH 8, containing RNaseA (20 μg/ml), DNAse (10 μg/ml), lysozyme (1 mg/ml), 10 mM imidazole as well as protein inhibitors and incubated for 1 h at 4°C with shaking. The cells were then lysed
with 3 min selleck products sonification, 40% amplitude and the supernatant obtained by centrifugation at 17000×g for 1 h at 4°C. The histidine-tag enabled Ni-NTA affinity column purification according to the user’s manual (Qiagen). Nonspecifically bound proteins were washed off the column with 50 mM phosphate, 300 mM NaCl, pH 8.0 buffer, containing 50 mM imidazole while colicin M was subsequently eluted with the same buffer containing Ketotifen 300 mM imidazole. The colicin-M-containing fractions, as established by 10% SDS-PAGE, were then dialyzed against 5 mM phosphate buffer, pH 7.3, centrifuged at 17,000× g at 4°C for 30 min, and stored at −80°C. Colicin M purity was verified by SDS-PAGE (see Additional file 4: Figure S3), and (a concentration of 3.4 mg/ml) protein concentrations were determined using bicinchoninic acid protein assay kits (Pierce) and a Nanodrop ND 1000 spectrophotometer (Thermo Scientific). Finally colicin M was stored at −80°C. Growth conditions The agar dilution method (National Committee for Clinical Laboratory Standards, 2000) was used to determine the minimal inhibitory concentration (MIC) of colicin M 50 ng/ml. For this purpose, an overnight culture of E. coli MG1655  was diluted 1:625 in LB broth and grown at 37°C with aeration to an OD600 0.6 when the culture was divided into several parts. One part served as a control while the other parts were treated with various concentrations of colicin M.