PMID:3041376
| Citation |
Bhattacharyya, D, Tano, K, Bunick, GJ, Uberbacher, EC, Behnke, WD and Mitra, S (1988) Rapid, large-scale purification and characterization of 'Ada protein' (O6 methylguanine-DNA methyltransferase) of E. coli. Nucleic Acids Res. 16:6397-410 |
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| Abstract |
The E. coli Ada protein (O6-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 (E280 nm (1%)) 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 O6-methylguanine in DNA. Its reaction with O6-methylguanine in a synthetic DNA has a second-order rate constant of 1.1 x 10(9) M-1 min-1 at O degree C. Both the native form and the protein methylated at Cys-69 are monomeric. The CD spectrum suggests a low alpha-helical content and the radius of gyration of 23 A 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. |
| Links | |
| Keywords |
Circular Dichroism; Cloning, Molecular; Escherichia coli/enzymology; Isoelectric Point; Kinetics; Methyltransferases/genetics; Methyltransferases/isolation & purification; Methyltransferases/metabolism; O(6)-Methylguanine-DNA Methyltransferase; Peptide Mapping; Plasmids; Protein Conformation; Spectrophotometry, Ultraviolet |
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