Recombinant Reconstitution of Sumoylation Reactions In Vitro

  • Annette Flotho
  • Andreas Werner
  • Tobias Winter
  • Andrea S. Frank
  • Heidi Ehret
  • Frauke MelchiorEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 832)


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.

Key words

SUMO SUMO E1-activating enzyme Aos1/Uba2 SUMO E2-conjugating enzyme Ubc9 SUMO E3 ligase Recombinant SUMO substrates In vitro sumoylation assay 



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).


  1. 1.
    Geiss-Friedlander R, Melchior F (2007) Concepts in sumoylation: a decade on. Nat Rev Mol Cell Biol 8:947–956.PubMedCrossRefGoogle Scholar
  2. 2.
    Gareau JR, Lima CD (2010) The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition. Nat Rev Mol Cell Biol 11:861–871.PubMedCrossRefGoogle Scholar
  3. 3.
    Mencia M, de Lorenzo V (2004) Functional transplantation of the sumoylation machinery into Escherichia coli. Protein Expr Purif 37:409–418.PubMedCrossRefGoogle Scholar
  4. 4.
    Uchimura Y, Nakao M, Saitoh H (2004) Generation of SUMO-1 modified proteins in E. coli: towards understanding the biochemistry/structural biology of the SUMO-1 pathway. FEBS Lett 564:85–90.PubMedCrossRefGoogle Scholar
  5. 5.
    Langereis MA, Rosas-Acosta G, Mulder K, Wilson VG (2007) Production of sumoylated proteins using a baculovirus expression system. J Virol Methods 139:189–194.PubMedCrossRefGoogle Scholar
  6. 6.
    Desterro JM, Rodriguez MS, Hay RT (1998) SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation. Mol Cell 2:233–239.PubMedCrossRefGoogle Scholar
  7. 7.
    Werner A, Moutty MC, Moller U, Melchior F (2009) Performing in vitro sumoylation reactions using recombinant enzymes. Methods Mol Biol 497:187–199.PubMedCrossRefGoogle Scholar
  8. 8.
    Pichler A, Knipscheer P, Saitoh H et al (2004) The RanBP2 SUMO E3 ligase is neither HECT- nor RING-type. Nat Struct Mol Biol 11:984–991.PubMedCrossRefGoogle Scholar
  9. 9.
    Reverter D, Lima CD (2005) Insights into E3 ligase activity revealed by a SUMO-RanGAP1-Ubc9-Nup358 complex. Nature 435:687–692.PubMedCrossRefGoogle Scholar
  10. 10.
    Seeler JS, Marchio A, Sitterlin D et al (1998) Interaction of SP100 with HP1 proteins: a link between the promyelocytic leukemia-associated nuclear bodies and the chromatin compartment. Proc Natl Acad Sci U S A 95:7316–7321.PubMedCrossRefGoogle Scholar
  11. 11.
    Pichler A, Knipscheer P, Oberhofer E et al (2005) SUMO modification of the ubiquitin-conjugating enzyme E2-25 K. Nat Struct Mol Biol 12:264–269.PubMedCrossRefGoogle Scholar
  12. 12.
    Schmidt D, Muller S (2002) Members of the PIAS family act as SUMO ligases for c-Jun and p53 and repress p53 activity. Proc Natl Acad Sci U S A 99:2872–2877.PubMedCrossRefGoogle Scholar
  13. 13.
    Meulmeester E, Kunze M, Hsiao HH et al (2008) Mechanism and consequences for paralog-specific sumoylation of ubiquitin-specific protease 25. Mol Cell 30:610–619.PubMedCrossRefGoogle Scholar
  14. 14.
    Zhu J, Zhu S, Guzzo CM et al (2008) Small ubiquitin-related modifier (SUMO) binding determines substrate recognition and paralog-selective SUMO modification. J Biol Chem 283:29405–29415.PubMedCrossRefGoogle Scholar
  15. 15.
    Fernandez-Miranda G, de Castro IP, Carmena M et al (2010) SUMOylation modulates the function of Aurora-B kinase. J Cell Sci 123:2823–2833.PubMedCrossRefGoogle Scholar
  16. 16.
    Hietakangas V, Anckar J, Blomster HA et al (2006) PDSM, a motif for phosphorylation-dependent SUMO modification. Proc Natl Acad Sci U S A 103:45–50.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Annette Flotho
    • 1
  • Andreas Werner
    • 1
  • Tobias Winter
    • 1
  • Andrea S. Frank
    • 1
  • Heidi Ehret
    • 1
  • Frauke Melchior
    • 1
    Email author
  1. 1.Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH AllianceHeidelbergGermany

Personalised recommendations