Overexpression of the wild-type gene coding for Escherichia coli DNA adenine methylase (dam).
The gene coding for Escherichia coli dam methylase was isolated from a dam+ K12 strain by the PCR method. The gene was subcloned into an overexpression vector under the control of the strong lambda PL promoter. The resultant construct produced the dam methylase at about 20% of total cellular protein. Purification of the protein was achieved with two chromatography columns and yielded 6 mg of pure methylase per gram cell paste. The methylase readily methylates the synthetic dodecamer GACTGATCAGTC containing its recognition sequence (underlined). It also methylates a synthetic dodecamer containing the EcoRV recognition sequence GATATC. However, methyl transfer is to the second adenine in the EcoRV sequence.
Nwosu
V U
VU
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
D001483:Base Sequence; D002845:Chromatography; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D008745:Methylation; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics* / Q000302:isolation & purification; D013379:Substrate Specificity
D004269:DNA, Bacterial; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases