SUMO Protocols pp 241-251 | Cite as

An In Vitro FRET-Based Assay for the Analysis of SUMO Conjugation and Isopeptidase Cleavage

  • Nicolas Stankovic-Valentin
  • Lukasz Kozaczkiewicz
  • Katja Curth
  • Frauke Melchior
Part of the METHODS IN MOLECULAR BIOLOGY™ book series (MIMB, volume 497)


To measure rates of sumoylation and isopeptidase cleavage in vitro, we developed an enzyme assay that is based on fl uorescence resonance energy transfer (FRET). FRET is a process by which the excited state energy of a fl uorescent donor molecule is transferred to an acceptor molecule. Effi cient energy transfer requires very close proximity, and can therefore be used as a read-out for covalent and non-covalent protein interactions. The assay described here uses bacterially expressed and purifi ed YFP-SUMO-1 and CFP-RanGAP1 as model substrates that are covalently coupled in the presence of recombinant SUMO E1 and E2 enzymes and ATP. Reactions of 25 μl volume, set up in 384-wells plates, give suffi cient signal for analysis. Consequently, this assay requires very low amounts of recombinant proteins and allows measurement of time courses in high-throughput format.

Key words

SUMO E1 activating enzyme E2 conjugating enzyme Aos1/Uba2 Ubc9 yellow fluorescent protein fluorescence resonance energy transfer FRET isopeptidase 



We would like to acknowledge previous and current lab members for contributing to the assay development, for sharing reagents and for many stimulating discussions. Funding by the EU (Rubicon and UbiRegulator), and fellowships by the Fondation pour la Recherche Médicale (to NS) and the Niedersachsen Lich-tenberg Program (to LK) are gratefully acknowledged.


  1. 1.
    Bossis, G., Chmielarska, K., Gartner, U., Pichler, A., Stieger, E., and Melchior, F. (2005) A fluorescence resonance energy transfer-based assay to study SUMO modification in solution. Methods Enzymol 398, 20–32.PubMedCrossRefGoogle Scholar
  2. 2.
    Piston, D. W., and Kremers, G. J. (2007) Fluorescent protein FRET: the good, the bad and the ugly. Trends Biochem Sci 32, 407–414.PubMedCrossRefGoogle Scholar
  3. 3.
    Tsien, R. Y. (1998) The green fluorescent protein. Annu Rev Biochem 67, 509–544.PubMedCrossRefGoogle Scholar
  4. 4.
    Mahajan, R., Gerace, L., and Melchior, F. (1998) Molecular characterization of the SUMO-1 modification of RanGAP1 and its role in nuclear envelope association. J Cell Biol 140, 259–270.PubMedCrossRefGoogle Scholar
  5. 5.
    Matunis, M. J., Wu, J., and Blobel, G. (1998) SUMO-1 modification and its role in targeting the Ran GTPase-activating protein, RanGAP1, to the nuclear pore complex. J Cell Biol 140, 499–509.PubMedCrossRefGoogle Scholar
  6. 6.
    Pichler, A., Gast, A., Seeler, J. S., Dejean, A., and Melchior, F. (2002) The nucleop-orin RanBP2 has SUMO1 E3 ligase activity. Cell 108, 109–120.PubMedCrossRefGoogle Scholar
  7. 7.
    Bossis, G., and Melchior, F. (2006) Regulation of SUMOylation by reversible oxidation of SUMO conjugating enzymes. Mol Cell 21, 349–357.PubMedCrossRefGoogle Scholar
  8. 8.
    Bailey, D., and O'Hare, P. (2004) Characterization of the localization and proteolytic activity of the SUMO-specific protease, SENP1. J Biol Chem 279, 692–703.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Nicolas Stankovic-Valentin
    • 1
  • Lukasz Kozaczkiewicz
    • 1
  • Katja Curth
    • 1
  • Frauke Melchior
    • 1
  1. 1.Department of Biochemistry IFaculty of Medicine, Georg-August University of GöttingenGermany

Personalised recommendations