Generating protein-linked and protein-free mono-, oligo-, and poly(ADP-ribose) in vitro

Ken Yu Lin, Dan Huang, W. Lee Kraus

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages91-108
Number of pages18
DOIs
StatePublished - Jan 1 2018
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume1813
ISSN (Print)1064-3745

Fingerprint

Poly Adenosine Diphosphate Ribose
Adenosine Diphosphate Ribose
Adenosine Diphosphate
Poly(ADP-ribose) Polymerases
ADP Ribose Transferases
Proteins
Baculoviridae
Enzymes
Post Translational Protein Processing
NAD
Insects
In Vitro Techniques
oligo adenosine diphosphoribose
Chromatography
Epitopes
DNA

Keywords

  • ADP-ribose (ADPR)
  • ADP-ribosylation
  • ADP-ribosyltransferase (ART)
  • ADPR binding domain (ARBD)
  • Automodification
  • Mono(ADP-ribosyl)ation (MARylation)
  • Nicotinamide adenosine dinucleotide (NAD)
  • Oligo(ADP-ribosyl)ation (OARylation)
  • Poly(ADP-ribose) polymerase (PARP)
  • Poly(ADP-ribosyl)ation (PARylation)
  • Posttranslational modification (PTM)

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Lin, K. Y., Huang, D., & Lee Kraus, W. (2018). Generating protein-linked and protein-free mono-, oligo-, and poly(ADP-ribose) in vitro. In Methods in Molecular Biology (pp. 91-108). (Methods in Molecular Biology; Vol. 1813). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-8588-3_7

Generating protein-linked and protein-free mono-, oligo-, and poly(ADP-ribose) in vitro. / Lin, Ken Yu; Huang, Dan; Lee Kraus, W.

Methods in Molecular Biology. Humana Press Inc., 2018. p. 91-108 (Methods in Molecular Biology; Vol. 1813).

Research output: Chapter in Book/Report/Conference proceedingChapter

Lin, KY, Huang, D & Lee Kraus, W 2018, Generating protein-linked and protein-free mono-, oligo-, and poly(ADP-ribose) in vitro. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 1813, Humana Press Inc., pp. 91-108. https://doi.org/10.1007/978-1-4939-8588-3_7
Lin KY, Huang D, Lee Kraus W. Generating protein-linked and protein-free mono-, oligo-, and poly(ADP-ribose) in vitro. In Methods in Molecular Biology. Humana Press Inc. 2018. p. 91-108. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-8588-3_7
Lin, Ken Yu ; Huang, Dan ; Lee Kraus, W. / Generating protein-linked and protein-free mono-, oligo-, and poly(ADP-ribose) in vitro. Methods in Molecular Biology. Humana Press Inc., 2018. pp. 91-108 (Methods in Molecular Biology).
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N2 - 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.

AB - 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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KW - Poly(ADP-ribosyl)ation (PARylation)

KW - Posttranslational modification (PTM)

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