Escherichia coli dnaB Protein

The purification of the Escherichia coli dnaB protein by affinity chromatography on nucleotides bound to agarose is described. The dnaB protein, which contains an associated ribonucleoside triphosphatase activity (Wickner, S., Wright, M., and Hurwitz, J. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 783-787) binds to immobilized ATP, ADP, and UDP, but not to AMP. The type of linkage of ATP to agarose influences the adsorption, elution, and purification of the enzyme. Optimal purification is achieved using ATP bound to agarose via its oxidized ribose moiety. By this means, the dnaB protein can be obtained at least 95% electrophoretically pure after only three purification steps. The enzyme can be eluted from immobilized nucleoside-5’-diand -triphosphates by ATP, ADP, and pyrophosphate, but not by AMP or orthophosphate. ADP and pyrophosphate, as well as the substrate ATP in high concentration are at the same time inhibitors of the ribonucleoside triphosphatase. The dnaB complementing and ribonucleoside triphosphatase activities could not be separated from each other by affinity chromatography, supporting the finding of others that they both reside on the same protein complex, namely a dnaB multimer. The results indicate that the dnaB protein binds to immobilized nucleotides by means of its ribonucleoside triphosphatase, and that at least the pyrophosphate moiety is essential for adsorption as well as elution of the enzyme.