PMID:2524489

Citation	Lohman, TM, Chao, K, Green, JM, Sage, S and Runyon, GT (1989) Large-scale purification and characterization of the Escherichia coli rep gene product. J. Biol. Chem. 264:10139-47
Abstract	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 lambda PL promoter (Colasanti, J., and Denhardt, D. T. (1987) Mol. Gen. Genet. 209, 382-390). The purification procedure results in greater than 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 epsilon 280 = 1.16 +/- 0.04 ml mg-1 cm-1 (8.47 +/- 0.28 X 10(4) M-1 cm-1) in 10 mM Tris (pH 7.5), 20% (v/v) glycerol, 0.10 M NaCl at 25 degrees 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 degrees 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 degrees C, in the presence of 20% glycerol and greater than or equal to 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 approximately 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 greater than acetate greater than 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.
Links	PubMed
Keywords	Adenosine Triphosphatases/genetics; Adenosine Triphosphatases/isolation & purification; Adenosine Triphosphatases/metabolism; Chromatography, Affinity; Chromosomes, Bacterial; DNA Helicases; DNA Replication; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/growth & development; Escherichia coli Proteins; Genes; Genes, Bacterial; Kinetics
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ECOLI:REP		GO:0003678: DNA helicase activity	ECO:0000247:	PMID:2524489	F		Figure 4	complete CACAO 4486

References

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