Engineering a high-affinity methyl-CpG-binding protein

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 v...

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Published inNucleic acids research Vol. 34; no. 13; p. e96
Main Authors Jørgensen, Helle F., Adie, Karen, Chaubert, Pascal, Bird, Adrian P.
Format Journal Article
LanguageEnglish
Published England Oxford University Press 01.01.2006
Oxford Publishing Limited (England)
Subjects
Animals
Cell Line
CpG Islands
DNA Methylation
DNA-Binding Proteins - chemistry
DNA-Binding Proteins - genetics
DNA-Binding Proteins - metabolism
Indicators and Reagents
Methods Online
Mice
Protein Engineering
Protein Structure, Tertiary
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
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Summary: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.
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To whom correspondence should be addressed at The Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Mayfield Road, Edinburgh EH9 3JR, UK. Tel: +44 131 650 8695; Fax: +44 131 650 5379; Email: A.bird@ed.ac.uk
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Present address: Helle F. Jørgensen, Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK
Correspondence may also be addressed to Helle F. Jørgensen. Tel: +44 208 383 8286; Fax: +44 208 383 8338; Email: helle.jorgensen@csc.mrc.ac.uk
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkl527