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• W2343972123 endingPage "e0154207" @default.
• W2343972123 startingPage "e0154207" @default.
• W2343972123 abstract "The ε-amino group of lysine residues may be mono-, di- or tri-methylated by protein lysine methyltransferases. In the past few years it has been highly considered that methylation of both histone and non-histone proteins has fundamental role in development and progression of various human diseases. Thus, the establishment of tools to study lysine methylation that will distinguish between the different states of methylation is required to elucidate their cellular functions. The 3X malignant brain tumor domain (3XMBT) repeats of the Lethal(3)malignant brain tumor-like protein 1 (L3MBTL1) have been utilized in the past as an affinity reagent for the identification of mono- and di-methylated lysine residues on individual proteins and on a proteomic scale. Here, we have utilized the 3XMBT domain to develop an enzyme-linked immunosorbent assay (ELISA) that allows the high-throughput detection of 3XMBT binding to methylated lysines. We demonstrated that this system allows the detection of methylated peptides, methylated proteins and PKMT activity on both peptides and proteins. We also optimized the assay to detect 3XMBT binding in crude E. coli lysates which facilitated the high throughput screening of 3XMBT mutant libraries. We have utilized protein engineering tools and generated a double site saturation 3XMBT library of residues 361 and 411 that were shown before to be important for binding mono and di-methylated substrates and identified variants that can exclusively recognize only di-methylated peptides. Together, our results demonstrate a powerful new approach that will contribute to deeper understanding of lysine methylation biology and that can be utilized for the engineering of domains for specific binders of other post-translational modifications." @default.
• W2343972123 created "2016-06-24" @default.
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• W2343972123 date "2016-04-25" @default.
• W2343972123 modified "2023-09-27" @default.
• W2343972123 title "Engineering of Methylation State Specific 3xMBT Domain Using ELISA Screening" @default.
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• W2343972123 doi "https://doi.org/10.1371/journal.pone.0154207" @default.
• W2343972123 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4844143" @default.
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• W2343972123 hasPublicationYear "2016" @default.
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