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Total chemical synthesis of histones and their analogs, assisted by native chemical ligation and palladium complexes

Nature Protocols volume 12, pages 22932322 (2017) | Download Citation

Abstract

Chemical synthesis of histones allows precise control of the installation of post-translational modifications via the coupling of derivatized amino acids. Shortcomings of other approaches for obtaining modified histones for epigenetic studies include heterogeneity of the obtained product and difficulties in incorporating multiple modifications on the same histone. In this protocol, unprotected peptide fragments are prepared by Fmoc solid-phase synthesis and coupled in aqueous buffers via native chemical ligation (NCL; in NCL, a peptide bond is formed between a peptide with an N-terminal Cys and another peptide having a C-terminal thioester). This task is challenging, with obstacles relating to the preparation and ligation of hydrophobic peptides, as well as the requirement for multiple purification steps due to protecting-group manipulations during the polypeptide assembly process. To address this, our approach uses an easily removable solubilizing tag for the synthesis and ligation of hydrophobic peptides, as well as a more efficient and better-yielding method to remove Cys-protecting groups that uses palladium chemistry (specifically [Pd(allyl)Cl]2 and PdCl2 complexes). The utility of this approach is demonstrated in the syntheses of ubiquitinated H2B at Lys34, phosphorylated H2A at Tyr57 and unmodified H4. Each of these analogs can be prepared in milligram quantities within 20–30 d.

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Acknowledgements

A.B. is a Neubauer Professor and a Taub Fellow supported by the Taub Foundation. S.K.M. thanks Israel's Council of Higher Education for a fellowship under the PBC program. M.J. thanks the Israel Council of Higher Education for a fellowship under his PhD program.

Author information

Affiliations

  1. Schulich Faculty of Chemistry, Technion-Israel Institute of Technology, Haifa, Israel.

    • Suman Kumar Maity
    • , Muhammad Jbara
    • , Guy Mann
    • , Guy Kamnesky
    •  & Ashraf Brik

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Contributions

S.K.M., M.J., G.M. and G.K. performed the experiments, compound characterization and data analysis. All the authors discussed the experimental design and contributed to writing the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Ashraf Brik.

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https://doi.org/10.1038/nprot.2017.049

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