Abstract
Sumoylation of proteins in vitro has evolved as an indispensable tool for the functional analysis of this post-translational modification. In this article we present detailed protocols for bacterial production of mammalian proteins necessary to perform in vitro sumoylation reactions, namely the E1 activating enzyme Aos1/Uba2 (SAE1/SAE2), the E2 conjugating enzyme Ubc9, SUMO-1 (identical protocols can be used for SUMO-2/3), and the catalytic domain of the E3 ligase RanBP2/Nup358. Two alternative procedures are described for the E1 enzyme, one depending on co-expression of His-Aos1 and untagged Uba2, and a second protocol for separate expression of His-Aos1 and Uba2-His and subsequent reconstitution of the active dimer. Two example conditions for in vitro sumoylation of RanGAP1 and Sp100 in the absence or presence of the SUMO E3 ligase RanBP2, respectively, are provided. Both protocols can be adapted easily to test in vitro conjugation of other target proteins and/or E3 ligases.
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References
Geiss-Friedlander, R. and Melchior, F. (2007) Concepts in sumoylation: a decade on. Nat. Rev. Mol. Cell. Biol. 8, 947–956.
Tatham, M. H., Jaffray, E., Vaughan, O. A., Desterro, J. M., Botting, C. H., Naismith, J. H. and Hay, R. T. (2001) Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9. J. Biol. Chem. 276, 5368–5374.
Schmidt, D. and 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. USA 99, 2872–2877.
Johnson, E. S. and Gupta, A. A. (2001) An E3-like factor that promotes SUMO conjugation to the yeast septins. Cell 106, 735–744.
Chiu, Y. H., Sun, Q. and Chen, Z. J. (2007) E1-L2 activates both ubiquitin and FAT10. Mol. Cell 27, 1014–1023.
Pichler, A., Gast, A., Seeler, J. S., Dejean, A. and Melchior, F. (2002) The nucleoporin RanBP2 has SUMO1 E3 ligase activity. Cell 108, 109–120.
Sapetschnig, A., Rischitor, G., Braun, H., Doll, A., Schergaut, M., Melchior, F. and Suske, G. (2002) Transcription factor Sp3 is silenced through SUMO modification by PIAS1. Embo J. 21, 5206–5215.
Matunis, M. J., Coutavas, E. and Blobel, G. (1996) A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J. Cell Biol. 135, 1457–1470.
Mahajan, R., Delphin, C., Guan, T., Gerace, L. and Melchior, F. (1997) A small ubiqui-tin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell 88, 97–107.
Acknowledgments
he authors gratefully acknowledge Tina Lampe, Annette Flotho and all group members for sharing reagents and Frank Rhode for excellent technical assistance. Funding was obtained from the EU (Rubicon, UbiRegulator) and the DFG (SFB 523, GRK 521).
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Werner, A., Moutty, MC., Möller, U., Melchior, F. (2009). Performing In Vitro Sumoylation Reactions Using Recombinant Enzymes. In: Ulrich, H.D. (eds) SUMO Protocols. METHODS IN MOLECULAR BIOLOGY™, vol 497. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-566-4_12
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DOI: https://doi.org/10.1007/978-1-59745-566-4_12
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-934115-80-0
Online ISBN: 978-1-59745-566-4
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