CAT was purified from the soluble fraction of cell proteins by affinity chromatography (Zaidenzaig and Shaw, 1976). In principle, CAT is selectively captured by immobilized chloramphenicol after pouring of a complex mixture of proteins (such as the soluble fraction of an E. coli cell lysate) over the affinity column. Since CAT is bound in dynamic equilibrium with chloramphenicol, the protein can be eluted from this column by a chloramphenicol-containing buffer in a second step of the purification process.
Cell extracts were prepared as follows. Cells from a 100 ml overnight culture were collected by centrifugation at 3,800.g, and resuspended in 3 ml of sample buffer (50 mM Tris.HCl (pH 8.0), 2 mM EDTA, 0.1 mM 2-mercaptoethanol). To this suspension 1 ml lysozyme (4 mg/ml in sample buffer), 50 µl of a protease inhibitor (phenylmethylsulfonyl fluoride (100 mM; Boehringer), or PefablocÒ SC (100 mM; Boehringer)), and 50 µl DNaseI (1 mg/ml) were added. After 15 min of shaking at 30°C, the cells were lysed by 5 freeze-thaw cycles. The insoluble material was removed by centrifugation at 28,600.g for 30 min, followed by filtration over a 0.22 µm filter (Millipore, Bedford, MA). This cleared cell lysate was loaded onto a chloramphenicol-caproate agarose column (Sigma), equilibrated in washing buffer containing 50 mM Tris.HCl (pH 7.8), 1 M NaCl, 1 mM EDTA, 0.1 mM 2-mercaptoethanol for CATI, and 50 mM Tris.HCl (pH 7.8), 0.3 M NaCl, 1 mM EDTA, 0.1 mM 2-mercaptoethanol for CATIII. Bound CAT was washed with 10 column volumes of this buffer, and subsequently eluted with 7 column volumes of the same buffer, supplemented with 5 mM chloramphenicol. The affinity columns were regenerated with 5 column volumes of washing buffer. Sample and buffers were applied onto the columns using an FPLCÒ system (Pharmacia) at a flow rate of 0.5 ml/min. By this procedure up to 15 mg of CAT could be obtained per 100 ml of starting cell suspension. The purity of each enzyme preparation was assessed by SDS polyacrylamide gel electrophoresis (see below).
To desalt FPLC-derived protein samples, dialysis at 4°C was performed against 0.5 mM Tris.HCl (pH 7.5), 1 mM NaCl, followed by dialysis against water. Semi-permeable membranes (molecular weight cutoff £ 10 kDa) were pre-treated by boiling in 200 mM NaHCO3, 5 mM EDTA (Pohl, 1990). Small quantities of protein solutions (up to 1 ml) were desalted by ultracentrifugation with MicroconÔ microconcentrators (Amicon Inc., Beverly, MA). Desalted protein samples were stored at -70°C, or in lyophilized form.