Overexpression and rapid purification of Escherichia coli formamidopyrimidine-DNA glycosylase.
Formamidopyrimidine DNA glycosylase (Fpg) is a DNA glycosylase with an associated AP lyase activity. As a DNA repair enzyme, Fpg excises several modified bases from DNA associated with exposure to oxidizing agents such as free radicals. Experiments in many laboratories have been limited by the availability of the enzyme, and its production required at least a week of work to complete its purification. We have devised a new method that decreases the time and expense of purification of Fpg that should render this protein accessible to any laboratory. Fpg was subcloned into a gamma P(L) promoter-containing vector (pRE) and overproduced in the appropriate Escherichia coli host cells to about 25% of the total cellular protein. Fpg was purified to homogeneity in a simple two-step procedure with a 50% saving in time when compared to the previously known procedure. Comparative studies showed that the excision of 8-hydroxyguanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 4,6-diamino-5-formamidopyrimidine, and to a lesser extent, 8-hydroxyadenine was virtually identical for the Fpg purified using this method and for the Fpg purified by the original method. Therefore, this method should prove useful for a large number of laboratories and further research on oxidative DNA damage.
Reddy
Prasad
P
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA. prasad.reddy@nist.gov
Jaruga
Pawel
P
O'Connor
Tim
T
Rodriguez
Henry
H
Dizdaroglu
Miral
M
D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism; D002266:Carboxymethylcellulose Sodium / Q000737:chemistry; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular; D004249:DNA Damage; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D009609:Nitrous Oxide / Q000737:chemistry; D011743:Pyrimidines / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D011743:Pyrimidines; D011994:Recombinant Proteins; C071023:2,6-diamino-4-hydroxy-5-formamidopyrimidine; C002992:8-hydroxyadenine; C039544:4,6-diamino-5-N-formamidopyrimidine; C024829:8-hydroxyguanine; D006147:Guanine; D043382:DNA-Formamidopyrimidine Glycosylase; D000225:Adenine; D009609:Nitrous Oxide; D002266:Carboxymethylcellulose Sodium