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ADP-ribosylation and NAD+ Utilizing Enzymes pp 91–108Cite as

Generating Protein-Linked and Protein-Free Mono-, Oligo-, and Poly(ADP-Ribose) In Vitro

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1813))

Abstract

ADP-ribosylation is a covalent posttranslational modification of proteins that is catalyzed by various types of ADP-ribosyltransferase (ART) enzymes, including members of the poly(ADP-ribose) polymerase (PARP) family. ADP-ribose (ADPR) modifications can occur as mono(ADP-ribosyl)ation, oligo(ADP-ribosyl)ation, or poly(ADP-ribosyl)ation, depending on the particular ART enzyme catalyzing the reaction, as well as the specific reaction conditions. Understanding the biology of ADP-ribosylation requires facile and robust means of generating and detecting the modification in all of its forms. Here we describe how to generate protein-linked mono(ADP-ribose), oligo(ADP-ribose), and poly(ADP-ribose) (MAR, OAR, and PAR, respectively) in vitro as an automodification of PARPs 1 or 3. First, epitope-tagged PARP-1 (a PARP polyenzyme) and PARP-3 (a PARP monoenzyme) are expressed individually in insect cells using baculovirus expression vectors, and purified using immunoaffinity chromatography. Second, the purified recombinant PARPs are incubated individually in the presence of different concentrations of NAD+ (as a donor of ADPR groups) and sheared DNA (to activate their catalytic activities) resulting in various forms of auto-ADP-ribosylation. Third, the products are confirmed using ADPR detection reagents that can distinguish among MAR, OAR, and PAR. Finally, if desired, the OAR and PAR can be deproteinized. The protein-linked and free MAR, OAR, and PAR generated in these reactions can be used as standards, substrates, or binding partners in a variety of ADPR-related assays.

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Change history

  • 18 June 2019

    William Lee Kraus’s name was incorrectly published in Pubmed. It has now been corrected to read: Kraus WL.

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Acknowledgments

The PARP-related research in the Kraus lab is supported by grants from the National Institutes of Health, NIDDK (DK069710), the Cancer Prevention and Research Institute of Texas (CPRIT) (RP160319), and the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowments.

Author information

Author notes

  1. Ken Y. Lin and Dan Huang contributed equally to this work

Authors and Affiliations

  1. Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Ken Y. Lin, Dan Huang & W. Lee Kraus

  2. Division of Basic Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Ken Y. Lin, Dan Huang & W. Lee Kraus

  3. Department of Cardiovascular Diseases, Clinical Center of Human Gene Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, P.R. China

    Dan Huang

Authors
  1. Ken Y. Lin
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  2. Dan Huang
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Corresponding author

Correspondence to W. Lee Kraus .

Editor information

Editors and Affiliations

  1. Department of Biology, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA

    Paul Chang

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Lin, K.Y., Huang, D., Kraus, W.L. (2018). Generating Protein-Linked and Protein-Free Mono-, Oligo-, and Poly(ADP-Ribose) In Vitro. In: Chang, P. (eds) ADP-ribosylation and NAD+ Utilizing Enzymes. Methods in Molecular Biology, vol 1813. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8588-3_7

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  • DOI: https://doi.org/10.1007/978-1-4939-8588-3_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-8587-6

  • Online ISBN: 978-1-4939-8588-3

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