We report a procedure for the large-scale purification of the Escherichia coli Rep protein, a helicase that is involved in the replication of the E. coli chromosome as well as a number of single-stranded bacteriophages. The procedure starts with E. coli cells harboring an overproducing plasmid, pRepO, in which the E. coli rep gene is under transcriptional control of the inducible λP(L) promoter (Colasanti, J., and Denhardt, D.T. (1987) Mol. Gen. Genet. 209, 382-390). The purification procedure results in > 98% pure Rep protein, which is free of contaminating nuclease activity, with yields of 40-50 mg of Rep protein/50 g of induced MZ-1/pRepO cells. We also show that cell death occurs upon inducing such a large overproduction of the E. coli Rep protein in MZ-1/pRepO. The Rep protein purified by this procedure has high specific single-stranded DNA-dependent ATPase activity, as well as helicase activity, with an apparent 3' to 5' directionality. The extinction coefficient of purified E. coli Rep protein is ε280 = 1.16 ± 0.04 ml mg-1 cm-1 (8.47 ± 0.28 x 104 M-1 cm-1) in 10 mM Tris (pH 7.5), 20% (v/v) glycerol, 0.10 M NaCl at 25°C. The solubility properties of the purified Rep protein have been examined as a function of glycerol, NaCl, MgCl2, ATP, and ADP concentrations at 25 and 37°C (pH 7.5). Rep protein solubility decreases significantly with decreasing concentrations of glycerol and monovalent salt and increasing temperature; however, the presence of 1.5 mM ATP or ADP or MgCl2 at low NaCl concentrations increases the solubility. At 4°C, in the presence of 20% glycerol and ≥ 50 mM NaCl, the free Rep protein exists as a stable monomer under all conditions examined (± ATP and ± MgCl2). The single-stranded DNA-dependent ATPase activity decreases with increasing glycerol concentration, such that in 25% (v/v) glycerol it has ~40% of its activity as compared to solutions that contain no glycerol. The dependence of the single-stranded DNA-dependent ATPase activity on salt concentration for a series of monovalent salts indicates the presence of both cation and anion effects, with decreasing activity in the order glutamate > acetate > chloride. The ability to obtain highly purified E. coli Rep protein in large quantities with relative ease will greatly facilitate physical characterizations of the protein and its interactions with DNA.
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|State||Published - Jan 1 1989|