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Reconstitution of the Recombinant RanBP2 SUMO E3 Ligase Complex

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SUMO

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1475))

Abstract

One of the few proteins that have SUMO E3 ligase activity is the 358 kDa nucleoporin RanBP2 (Nup358). While small fragments of RanBP2 can stimulate SUMOylation in vitro, the physiologically relevant E3 ligase is a stable multi-subunit complex comprised of RanBP2, SUMOylated RanGAP1, and Ubc9. Here, we provide a detailed protocol to in vitro reconstitute the RanBP2 SUMO E3 ligase complex. With the exception of RanBP2, reconstitution involves untagged full-length proteins. We describe the bacterial expression and purification of all complex components, namely an 86 kDa His-tagged RanBP2 fragment, the SUMO E2-conjugating enzyme Ubc9, RanGAP1, and SUMO1, and we provide a protocol for quantitative SUMOylation of RanGAP1. Finally, we present details for the assembly and final purification of the catalytically active RanBP2/RanGAP1*SUMO1/Ubc9 complex.

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Acknowledgments

We are very grateful to H. Ehret, A. Frank, and K. Meese for excellent technical assistance, and acknowledge funding from the Deutsche Forschungsgemeinschaft for research on the RanBP2 complex (SFB523 TPA18, DFG SFB638 TPB8 and GRK 1188). The authors would like to acknowledge networking support by the PROTEOSTASIS action BM1307, supported by COST (European Cooperation in Science and Technology).

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Authors and Affiliations

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

    Tobias Ritterhoff, Hrishikesh Das, Yuqing Hao, Volkan Sakin, Annette Flotho, Andreas Werner & Frauke Melchior

  2. Department of Biochemistry, University of Washington, Seattle, Washington, USA

    Tobias Ritterhoff

  3. Department of Infectious Diseases and Virology, University Hospital of Heidelberg, Heidelberg, Germany

    Volkan Sakin

Authors
  1. Tobias Ritterhoff
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  2. Hrishikesh Das
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  3. Yuqing Hao
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  4. Volkan Sakin
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  5. Annette Flotho
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  6. Andreas Werner
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  7. Frauke Melchior
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Corresponding authors

Correspondence to Andreas Werner or Frauke Melchior .

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Editors and Affiliations

  1. INBIOMED, San Sebastian, Spain

    Manuel S. Rodriguez

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© 2016 Springer Science+Business Media New York

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Ritterhoff, T. et al. (2016). Reconstitution of the Recombinant RanBP2 SUMO E3 Ligase Complex. In: Rodriguez, M. (eds) SUMO. Methods in Molecular Biology, vol 1475. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6358-4_3

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  • DOI: https://doi.org/10.1007/978-1-4939-6358-4_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6356-0

  • Online ISBN: 978-1-4939-6358-4

  • eBook Packages: Springer Protocols

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