Open AccessPurification of theE.coliogt gene product to homogeneity and its rate of action onO6-methylguanine,O6-ethyiguanine andO4-methylthymine in dodecadeoxyribnucleotides
Author(s) -
Mark C. Wilkinson,
Philip M. Potter,
Lynn Cawkwell,
Panagiotis Georgiadis,
Dilip D. Patel,
Peter Swann,
Geoffrey P. Margison
Publication year - 1989
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/17.21.8475
Subject(s) - biology , microbiology and biotechnology , escherichia coli , gene , genetics
The E. coli gene ogt encodes the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (O6-AlkG ATase). The protein coding region of the gene was cloned into a multicopy expression vector to obtain high yields of the enzyme (approximately 0.2% of total protein) which was purified to apparent homogeneity by affinity, molecular exclusion and reverse-phase chromatography. Good correlation was found between the determined and predicted amino acid compositions. The ability of the purified protein to act on O6-methylguanine (O6-MeG), O6-ethylguanine (O6-EtG) and O4-methylthymine (O4-MeT) in self-complementary dodecadeoxyribonucleotides was compared to that of 19 kDa fragment of the related ada-protein. With both proteins the rate order was O6-MeG greater than O6-EtG greater than O4-MeT, however, the ogt protein was found to repair O6-MeG, O6-EtG and O4-Met, 1.1, 173 and 84 times, respectively, faster than the ada protein.