Nucleic Acids Research, 1988, Vol. 16, No. 14 6397-6410
© 1988
Articles |
Rapid, large-scale purification and characterization of ‘Ada protein’ (06 methylguanine-DNA methyltransferase) of E.coli
1Protein Engineering and Molecular Mutagenesis Program and University of Tennessee Graduate School of Biomedical Sciences Oak Ridge, TN 37831 2Protein Engineering and Molecular Mutagenesis Program and Biology Division, Oak Ridge National Laboratory Oak Ridge, TN 37831 3Protein Engineering and Molecular Mutagenesis Program and Solid State Division, Oak Ridge National Laboratory Oak Ridge, TN 37831 4Department of Biochemistry and Molecular Biology, University of Cincinnati College of Medicine Cincinnati, OH 45267, USA
*To whom correspondence should be addressed
Received March 1, 1988.
The E. coli Ada protein (06-methylguanine-DNA methyltransferase) has been purified using a high-level expression vector with a yield of about 3 mg per liter of E. coli culture. The 39-kDa protein has an extinction coefficient (E1%280nm) of 5.3. Its isoelectric point of 7.1 is lower than that predicted from the amino acid content. The homogeneous Ada protein is fully active as a methyl acceptor from 06-methylguanine in DNA. Its reaction with 06-methylguanine in a synthetic DNA has a second-order rate constant of 1.1 x 109 M–1 min–1 at 0°C. Both the native form and the protein methylated at Cys-69 are monomeric. The CD spectrum suggests a low 
-helical content and the radius of gyration of 23 Å indicates a compact, globular shape. The middle region of the protein is sensitive to a variety of proteases, including an endogenous activity in E. coli, suggesting that the protein is composed of N-terminal and C-terminal domains connected by a hinge region. E. coli B has a higher level of this protease than does K12.