Rapid, large-scale purification and characterization of ‘Ada protein’ (06methylguanine-DNA methyltransferase) ofE.coli
- 25 July 1988
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 16 (14) , 6397-6410
- https://doi.org/10.1093/nar/16.14.6397
Abstract
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 (E 1%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 × 10 9 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 α-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.Keywords
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