Abstract
The development of expressed protein ligation (EPL) widened the scope of questions that could be addressed by mechanistic biochemistry. Protein trans-splicing (PTS) relies on the same basic chemical principles, but utilizes split inteins to tracelessly ligate distinct peptide or polypeptide fragments together with native peptide bonds. Here we present a method to adapt PTS methodologies for their use in live cells, in order to deliver synthetic or native histone modifications. As an example, we provide a protocol to incorporate a small molecule fluorophore into chromatinized histones. The protocol should be easily adaptable to incorporate other modifications to chromatin in vivo.
Keywords:
Chromatin; In cellulo labeling; Split inteins; Synthetic biology.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Blotting, Western / methods
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Cell Fractionation / methods
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Cell-Penetrating Peptides / chemistry
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Cell-Penetrating Peptides / metabolism
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Chromatin / chemistry
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Chromatography, High Pressure Liquid / methods
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Chromatography, Reverse-Phase / methods
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Disulfides / chemistry
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Fluorescent Dyes / chemistry
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Gene Expression
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HEK293 Cells
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Histones / chemistry*
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Histones / metabolism
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Humans
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Inteins
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Microscopy, Confocal
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Nuclear Proteins / metabolism
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Protein Splicing*
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Solid-Phase Synthesis Techniques / methods*
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Synthetic Biology / methods*
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Transfection
Substances
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Cell-Penetrating Peptides
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Chromatin
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Disulfides
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Fluorescent Dyes
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Histones
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Nuclear Proteins
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Recombinant Proteins