Rapid, large-scale purification and characterization of 'Ada protein' (O6 methylguanine-DNA methyltransferase) of E. coli.
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.
D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D007526:Isoelectric Point; D007700:Kinetics; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D019853:O(6)-Methylguanine-DNA Methyltransferase; D010449:Peptide Mapping; D010957:Plasmids; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase