title: Engineering a high-affinity methyl-CpG-binding protein.
abstract: Core members of the MBD protein family (MeCP2, MBD1, MBD2 and MBD4) share a methyl-CpG-binding domain that has a specific affinity for methylated CpG sites in double-stranded DNA. By multimerizing the MDB domain of Mbd1, we engineered a poly-MBD protein that displays methyl-CpG-specific binding in vitro with a dissociation constant that is >50-fold higher than that of a monomeric MBD. Poly-MBD proteins also localize to methylated foci in cells and can deliver a functional domain to reporter constructs in vivo. We propose that poly-MBD proteins are sensitive reagents for the detection of DNA methylation levels in isolated native DNA and for cytological detection of chromosomal CpG methylation.
authors.affiliation: The Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK. helle.jorgensen@csc.mrc.ac.uk
authors.forename: Helle F
authors.initials: HF
authors.lastname: Jørgensen
authors.forename: Karen
authors.initials: K
authors.lastname: Adie
authors.forename: Pascal
authors.initials: P
authors.lastname: Chaubert
authors.forename: Adrian P
authors.initials: AP
authors.lastname: Bird
mesh_terms: D000818:Animals; D002460:Cell Line; D018899:CpG Islands; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007202:Indicators and Reagents; D051379:Mice; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D007202:Indicators and Reagents; D011994:Recombinant Proteins