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Purification of TET Proteins

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TET Proteins and DNA Demethylation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2272))

Abstract

The 5-methylcytosine (5mC) oxidation pathway mediated by TET proteins involves step-wise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC can be removed from DNA by base excision repair and the completion of this pathway results in “demethylation” of 5mC by converting the modified base back into cytosine. In vitro studies with TET proteins aimed at analyzing their DNA substrate specificities and their activity within defined chromatin templates are relatively limited. Here we describe purification methods for mammalian TET proteins based on expression in insect cells or in 293T cells. We also briefly summarize a method that can be used to monitor 5-methylcytosine oxidase activity of the purified TET proteins in vitro.

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Author information

Authors and Affiliations

  1. Center for Epigenetics, Van Andel Institute, Grand Rapids, MI, USA

    Zhijun Huang, Jiyoung Yu, Jennifer Johnson, Seung-Gi ** & Gerd P. Pfeifer

  2. Asan Medical Center, University of Ulsan, College of Medicine, Seoul, South Korea

    Jiyoung Yu

Authors
  1. Zhijun Huang
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  2. Jiyoung Yu
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  3. Jennifer Johnson
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  4. Seung-Gi **
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  5. Gerd P. Pfeifer
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Corresponding author

Correspondence to Gerd P. Pfeifer .

Editor information

Editors and Affiliations

  1. Genomics and Epigenetics Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia

    Ozren Bogdanovic

  2. Radboud University, Nijmegen, The Netherlands

    Michiel Vermeulen

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Huang, Z., Yu, J., Johnson, J., **, SG., Pfeifer, G.P. (2021). Purification of TET Proteins . In: Bogdanovic, O., Vermeulen, M. (eds) TET Proteins and DNA Demethylation. Methods in Molecular Biology, vol 2272. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1294-1_13

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  • DOI: https://doi.org/10.1007/978-1-0716-1294-1_13

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1293-4

  • Online ISBN: 978-1-0716-1294-1

  • eBook Packages: Springer Protocols

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