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Ubiquitin Family Modifiers and the Proteasome pp 93–110Cite as

Recombinant Reconstitution of Sumoylation Reactions In Vitro

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 832))

Abstract

Reconstituting posttranslational modification with SUMO in vitro is an essential tool in the analysis of sumoylation. In this article, we provide detailed protocols that allow to set up and perform sumoylation reactions using a purified recombinant sumoylation machinery. The protocols include purification of the SUMO E1 enzyme His-Aos1/Uba2, untagged E2 enzyme Ubc9, untagged SUMO, and the RanBP2 E3 ligase fragment IR1 + M. Using these components, we provide step-by-step instructions to set up sumoylation reactions. Two established SUMO model substrates, His-RanGAPtail and HisYFP-Sp100, complement the described tool box; these proteins serve as positive controls in E3 ligase-independent and -dependent sumoylation reactions and are valuable instruments to adjust the reaction conditions if necessary.

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Acknowledgments

We are grateful to Nicolas Stankovic-Valentin for critical reading of the manuscript, Anja Schreieck for excellent assistance in establishing the improved version of the E1 purification procedure, Tina Lampe for cloning HisYFP-Sp100, and all lab members for sharing reagents and their experience. The group acknowledges funding from the DFG (SFB 523, GRK 1188) and the EU (NoE Rubicon).

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Authors and Affiliations

  1. Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, Heidelberg, 69120, Germany

    Annette Flotho, Andreas Werner, Tobias Winter, Andrea S. Frank, Heidi Ehret & Frauke Melchior

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  1. Annette Flotho
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  2. Andreas Werner
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  3. Tobias Winter
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  4. Andrea S. Frank
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  5. Heidi Ehret
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  6. Frauke Melchior
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Correspondence to Frauke Melchior .

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Editors and Affiliations

  1. Institut für Genetik, Universität Köln, Zülpicher Str. 47, Köln, 50674, Germany

    R. Jürgen Dohmen

  2. Fak. Biologie, Universität Konstanz, Konstanz, 78457, Germany

    Martin Scheffner

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Flotho, A., Werner, A., Winter, T., Frank, A.S., Ehret, H., Melchior, F. (2012). Recombinant Reconstitution of Sumoylation Reactions In Vitro. In: Dohmen, R., Scheffner, M. (eds) Ubiquitin Family Modifiers and the Proteasome. Methods in Molecular Biology, vol 832. Humana Press. https://doi.org/10.1007/978-1-61779-474-2_5

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  • DOI: https://doi.org/10.1007/978-1-61779-474-2_5

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-473-5

  • Online ISBN: 978-1-61779-474-2

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