Protein expression and purification A mL overnight culture o
Protein expression and purification. A 3 mL overnight culture of E. coli BL21 (DE3) pLysS pGEX-6P-3-A1S_0222 was grown in Luria-Bertani (LB)-Medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, pH 7.4, 100 μg/mL Amp) at 37 °C and 160 rpm. The overnight culture was diluted 1:100 into 200 mL LB (supplemented with 100 μg/mL Amp) and cultured in a 2 L bottle-flask at 20 °C and 160 rpm for 5 h. Expression was induced by addition of 0.05 mM IPTG and cultures were incubated for 16 h at 20 °C and 160 rpm. Cells were then harvested by centrifugation (10,000 × g for 30 min at 4 °C) and the resulting cell pellets were frozen at −80 °C. Cell pellets were solubilized at 4 °C in 20 mL disruption buffer (300 mM NaCl, 1 mM DTT (1,4-dithiothreitol), 5 mM EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid), 1 mM EDTA (2,2′,2″,2‴-(ethane-1,2-diyldinitrilo) tetraacetic acid), 2 μL benzonase nuclease (250 U/μL), pH 7.4. The p-nitro-Cyclic Pifithrin-α were lysed by applying three passages through an EmulsiFlex-C3 homogenizer (Avestin). Lysates were then centrifuged at 20,000 × g for 50 min at 4 °C. The soluble lysate was then added to a GSTPrep™ FF 16/10 column (GE Healthcare Life Sciences). GST affinity chromatography. The GSTPrep™ FF 16/10 column (bed volume 20 mL) was equilibrated with 5 column volumes (CV) of GST binding buffer (300 mM NaCl, 1 mM DTT, 5 mM EGTA, 1 mM EDTA, 5% glycerol (v/v), pH 7.4). The soluble lysate was added to the column and washed with 10 CV of GST binding buffer and eluted with 7 CV of GST elution buffer (300 mM NaCl, 50 mM Tris, 1 mM DTT, 5 mM EGTA, 1 mM EDTA, 5% glycerol (v/v), 10 mM reduced l-glutathione (GSH reduced), pH 7.4). Eluted fractions were analyzed by SDS-PAGE and appropriate fractions were pooled. The GST fusion protein was then incubated for 16 h at 8 °C with 40 units PreScission protease per 14.52 mg recombinant A1S_0222 to cleave off the GST tag (26 kDa). After incubation the pooled fractions were centrifuged at 20,000 g for 20 min at 4 °C and diluted 1:4 in dilution buffer (50 mM Tris, 1 mM EDTA, 1 mM DTT, 5% glycerol (v/v)). Cation exchange chromatography (CEC). The HiTrap™ SP XL (GE Healthcare Life Sciences) column (bed volume 1 mL) was equilibrated with 10 column volumes (CV) CEC binding buffer (50 mM NaCl, 50 mM Tris, 1 mM DTT, 1 mM EDTA, 5% glycerol (v/v)). The protein solution was loaded on the column and the column washed with 5 CV CEC binding buffer and subsequently eluted with 30 CV in a linear gradient from 0% to 100% of CEC elution buffer (1000 mM NaCl, 50 mM Tris, 1 mM DTT, 1 mM EDTA, 5% glycerol (v/v)). Eluted fractions were analyzed by reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), pooled and loaded on a PD-10 desalting column. PD-10 desalting column. The PD-10 desalting (GE Healthcare Life Sciences) column was equilibrated with buffer B1 (150 mM NaCl, 10 mM Tris, 1 mM DTT and 5% glycerol (v/v), pH 7.4). Further steps were performed as described in the column manual using a gravity desalting protocol. The A1S_0222 protein was eluted with buffer B1. Pooled fractions were concentrated using the Vivaspin® concentrator with a molecular weight cutoff of 10,000 Da and a Hydrosart (HY) membrane (Sartorius Stedim Biotech). The purity of A1S_0222 was analyzed by reducing SDS-PAGE. Protein concentrations were determined using the Bradford method. Purified A1S_0222 was directly used for further analysis or stored at −80 °C. Confirmation of the A1S_0222 DNA methylation recognition site. To confirm the A1S_0222 DNA methylation recognition site, the 800 base-pair (bp) PCR product, Int1, consisting of the 5′-end of the a1s_0222 gene from A. baumannii ATCC 17978, was incubated with and without purified A1S_0222 at 37 °C for 1 h (3 μL Int1 (about 200 ng/μL), 2 μL B1 buffer, 4 μL of 800 μM S-adenosyl-l-methionine (SAM), 3 μL A1S_0222 (2.46 μg/μL), 8 μL RNase free water). Int1 was amplified with the oligonucleotides Int1-for: 5′-GGATCCGGATGAAATGATCAGTTATGTGGC-3′ and Int1-rev: 5′-CGCTCTAATGCTGTTTGTGTACG-3’. Samples of methylated and non-methylated Int1 DNA were used for Sanger DNA sequencing and analyzed at the Robert Koch-Institute sequencing lab (Berlin, Germany) on an ABI PRISM analyzer. The sequencing chromatograms were analyzed as described previously .