ArticleProtein engineering, design & selection : PEDS2015
Characterization and directed evolution of a methyl-binding domain protein for high-sensitivity DNA methylation analysis.
Article in Protein engineering, design & selection : PEDS, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
What it found
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
The trial behind it
Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
5 citing papers in PubMed, 12 citations in OpenAlex.
- Density Control over MBD2 Receptor-Coated Surfaces Provides Superselective Binding of Hypermethylated DNA.ACS applied materials & interfaces · 2022Article
- Modified Histone Peptides Linked to Magnetic Beads Reduce Binding Specificity.International journal of molecular sciences · 2022Article
- Structural Basis of MeCP2 Distribution on Non-CpG Methylated and Hydroxymethylated DNA.Journal of molecular biology · 2017Article
- Engineering affinity agents for the detection of hemi-methylated CpG sites in DNA.Molecular systems design & engineering · 2016Article
- MethylMeter(®): bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples.Epigenomics · 2016Article
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
Methyl-binding domain (MBD) family proteins specifically bind double-stranded, methylated DNA which makes them useful for DNA methylation analysis. We displayed three of the core members MBD1, MBD2 and MBD4 on the surface of Saccharomyces cerevisiae cells. Using the yeast display platform, we determined the equilibrium dissociation constant of human MBD2 (hMBD2) to be 5.9 ± 1.3 nM for binding to singly methylated DNA. The measured affinity for DNA with two methylated sites varied with the distance between the sites. We further used the yeast display platform to evolve the hMBD2 protein for improved binding affinity. Affecting five amino acid substitutions doubled the affinity of the wild-type protein to 3.1 ± 1.0 nM. The most prevalent of these mutations, K161R, occurs away from the DNA-binding site and bridges the N- and C-termini of the protein by forming a new hydrogen bond. The F208Y and L170R mutations added new non-covalent interactions with the bound DNA strand. We finally concatenated the high-affinity MBD variant and expressed it in Escherichia coli as a green fluorescent protein fusion. Concatenating the protein from 1× to 3× improved binding 6-fold for an interfacial binding application.
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Registered trials
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