Identifying and Validating Tankyrase Binders and Substrates: A Candidate Approach
| Autor: | Sebastian Guettler, Ian Collins, Katie Pollock, Michael Ranes |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Tankyrases Binding Sites 030102 biochemistry & molecular biology Plasma protein binding Telomere Biology Article 3. Good health Protein–protein interaction Poly ADP Ribosylation 03 medical and health sciences 030104 developmental biology Structural biology Biochemistry Protein purification biology.protein Animals Humans Ankyrin repeat Peptide sequence Polymerase Protein Binding |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493969920 |
| DOI: | 10.1007/978-1-4939-6993-7_28 |
| Popis: | The poly(ADP-ribose)polymerase (PARP) enzyme tankyrase (TNKS/ARTD5, TNKS2/ARTD6) uses its ankyrin repeat clusters (ARCs) to recognize degenerate peptide motifs in a wide range of proteins, thereby recruiting such proteins and their complexes for scaffolding and/or poly(ADP-ribosyl)ation. Here, we provide guidance for predicting putative tankyrase-binding motifs, based on the previously delineated peptide sequence rules and existing structural information. We present a general method for the expression and purification of tankyrase ARCs from Escherichia coli and outline a fluorescence polarization assay to quantitatively assess direct ARC-TBM peptide interactions. We provide a basic protocol for evaluating binding and poly(ADP-ribosyl)ation of full-length candidate interacting proteins by full-length tankyrase in mammalian cells. |
| Databáze: | OpenAIRE |
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