Dnase rnase

Bacterial soluble proteins (= cell-associated proteins) were prepared from xylose and OSX cultures (500 ml) harvested at late-log phase. Bacterial cell pellets were first washed 3 times in distilled water containing a protease inhibitor (Complete EDTA free protease inhibitor cocktail, Roche, France) and re-suspended in 1/25 volume of the same solution. Cells were broken by one passage at 2000 bars using a One Shot cell Disruptor (CellD, France). The obtained suspension was treated with 270 U endonucleases (Dnase + RNase, Sigma, France) for 30 min at ambient temperature. Unbroken cells were removed by centrifugation (23,000 g, 40 min, 4 °C). The supernatant was subsequently submitted to ultracentrifugation (100,000 g, 1 h, 4 °C) to obtain a final supernatant containing bacterial soluble proteins that were aliquoted and frozen. Before analysis, an aliquot of protein solution was mixed with 3 volumes of ice-cold acetone and kept at -20 °C for at least 2 h. After centrifugation (13,000 g, 30 min, 4 °C), the protein pellet was dried under vacuum for 5 min and solubilized in isoelectric focusing (IEF) buffer (7 M urea, 2 M thiourea, 4 % CHAPS, 0.2 % triton X100). The amount of proteins solubilised in IEF buffer was determined with the PlusOne™ 2-D Quant kit (Amersham, France) according to the supplier’s instructions.