RAPID, LARGE-SCALE PURIFICATION AND CHARACTERIZATION OF ADA PROTEIN (O-6 METHYLGUANINE-DNA METHYLTRANSFERASE) OF ESCHERICHIA-COLI

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 extincton 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 .times. 109 M-1 min-2 at 0.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 .ANG. 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.