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Preparation of Recombinant Histones and Widom 601 DNA for Reconstitution of Nucleosome Core Particles

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DNA-Protein Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2599))

Abstract

Expression and purification of individual histone proteins and amplification and purification of DNA are the initial steps toward reconstituting nucleosome core particles. Histone proteins are expressed in E. coli, extracted from inclusion bodies, and purified using ion-exchange chromatography. DNA containing the 147 base pair Widom 601 sequence is amplified in bacteria using a plasmid containing multiple copies of this strong nucleosome positioning sequence. Following alkaline lysis of bacteria, DNA is extracted using phenol and chloroform, released from the vector via restriction enzyme digestion, and purified in subsequent precipitation and ion-exchange chromatography steps. Here, we describe a combination of two protocols: one to express and purify recombinant human histones and the other to amplify and purify Widom 601 DNA.

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Acknowledgments

Research reported in this publication was supported in part by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM142594 and the Medical College of Wisconsin Research Affairs Committee. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Thanks to Drs. Karolin Luger, Catherine Musselman, and Michael Poirier for gifts of histone and Widom 601 plasmids.

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Authors and Affiliations

  1. Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, USA

    Emeleeta A. Paintsil & Emma A. Morrison

Authors
  1. Emeleeta A. Paintsil
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  2. Emma A. Morrison
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Correspondence to Emma A. Morrison .

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Editors and Affiliations

  1. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA

    Marcos Simoes-Costa

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Paintsil, E.A., Morrison, E.A. (2023). Preparation of Recombinant Histones and Widom 601 DNA for Reconstitution of Nucleosome Core Particles. In: Simoes-Costa, M. (eds) DNA-Protein Interactions. Methods in Molecular Biology, vol 2599. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2847-8_12

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  • DOI: https://doi.org/10.1007/978-1-0716-2847-8_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2846-1

  • Online ISBN: 978-1-0716-2847-8

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