Purification of theE.coliogt gene product to homogeneity and its rate of action onO6-methylguanine,O6-ethyiguanine andO4-methylthymine in dodecadeoxyribnucleotides

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Abstract
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 (˜ 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 > O6 -EtG > 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.