2 ml/min, with ice cooling. After washing with five column volumes of ice-cold binding buffer, proteins were eluted with 5 ml binding buffer containing 10 mM reduced glutathione (Sigma-Aldrich, USA), collecting 1.0 ml fractions. Protein selleckchem fractions were analyzed on 12%, 15% or 20% SDS-PAGE gels, using colloidal Coomassie Brilliant Blue G-250 staining (Bio-Rad, USA). Analogous procedures were used for recombinant Z. mobilis ATCC 29191 and CU1 Rif2 strains, except that cultures (800 ml) were grown in RM media containing 100 μg/ml Cm at 30°C to an OD600nm of ca. 1.5-2.0.
Cell cultures were incubated semi-aerobically Saracatinib datasheet or anaerobically (in pre-reduced RM medium) in an anaerobic chamber (Forma Anaerobic System, Thermo Fisher Scientific), using a gas mixture of 85% nitrogen, 10% carbon dioxide and 5% hydrogen; as indicated in the text. Cultures of wild type Z. mobilis ATCC 29191 or CU1 Rif2 were analogously used as negative controls. The mass of pelleted cells obtained from 800 ml cultures was routinely ca.
2.5-3 g. Respective pZ7-GST plasmid-based protein expression levels were estimated by comparing band intensities on SDS-PAGE gels with those of a dilution series of purified recombinant GST protein of known concentration. Individual protein bands were carefully excised find more using a sterile scalpel, and were analyzed by a combination of mass spectrometric methods: peptide mass fingerprinting (PMF) of tryptic fragments, and LC-MS/MS analysis and peptide sequencing (Proteomic Laboratory for Systems Biology Research, Baptist University of Hong Kong, Hong Kong
SAR). Analysis of pZ7C plasmid-based GST fusion protein expression by Western Blotting After resolution on 12% SDS-polyacrylamide gels, proteins present in the fractions eluted from GST-affinity columns were wet-transferred Etofibrate to polyvinylidene fluoride (PVDF) membranes using transfer buffer (25 mM Tris-HCl pH 8.3, 190 mM glycine, 20% methanol). Membranes were blocked using blocking buffer [Tris-buffered saline with Tween 20 (TBST) containing 5% non-fat milk powder] for 1 hour at room temperature. Membranes were incubated with anti-GST primary antibody (Sigma Aldrich, USA; cat. #G7781) in blocking buffer (1:2500 dilution) for 12 hours at 4°C. After washing three times with TBST, membranes were incubated with secondary antibody (HRP-linked anti-rabbit IgG; Cell Signaling Technology, USA; cat. #7074P) in blocking buffer (1:2500 dilution) for 2 hours at room temperature; before being washed three times in TBST. The membrane blots were visualized chemiluminescently using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Thermo Scientific, USA; cat. #34079), capturing images using a Bio-Rad ChemiDoc XRS instrument (Bio-Rad, USA). The plasmid sequences pZMO1A and pZMO7 were deposited to the NCBI GenBank database with the accession numbers NC_019198 and NC_019300, respectively. Results Native plasmids in Z. mobilis NCIMB 11163 The NCIMB 11163 strain of Z.