Amplification and purification of UvrA, UvrB, and UvrC proteins of Escherichia coli.
The UvrA, UvrB, and UvrC proteins of Escherichia coli are subunits of a DNA repair enzyme, ABC exci nuclease. In order to amplify these proteins, we have joined the artificial canonical promoter tac (Amann E., Brosius, J., and Ptashne, M. (1983) Gene (Amst.) 25, 167-178) to the uvr genes to obtain plasmids that express these genes under the control of the lac repressor. When cells carrying the tac-uvr plasmids are induced by the gratuitous lac inducer isopropyl-beta-D-galactoside the Uvr proteins are overproduced reaching a level of 10-20% of total cellular proteins after 6-8 h of induction. We have developed methods to purify all three Uvr proteins, UvrA, UvrB, and UvrC, in milligram quantities and to near homogeneity from these overproducing cells. The purified UvrA protein is an ATPase but UvrB and UvrC proteins are not. However, UvrB protein stimulates the ATPase activity of UvrA protein by a factor of 1.5 in the presence of double-stranded DNA and by a factor of about 2.6 in the presence of UV-irradiated DNA but not in the absence of DNA.
Thomas
D C
DC
Levy
M
M
Sancar
A
A
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000528:radiation effects; D004260:DNA Repair; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005784:Gene Amplification; D010957:Plasmids
D001426:Bacterial Proteins; D004247:DNA; D000251:Adenosine Triphosphatases