Abstract
The use of in vitro assays, such as glutathione S-transferase (GST) pull-downs, enables the study of complex cellular processes in a simplified form. Pull-down assays facilitate the discovery and detailed study of protein–protein interactions, which can then be extrapolated to the cellular environment. Here, we describe the expression, purification and use of a multi-SUMO platform to identify SUMO-interacting proteins. This SUMO-platform can be easily expressed and purified from bacterial cells for use as baits in pull-down assays. This methodology facilitates the discovery of novel SUMO-binding proteins or further characterization of SUMO with known binding partners.
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References
Flotho A, Melchior F (2013) Sumoylation: a regulatory protein modification in health and disease. Annu Rev Biochem 82:357–385
Song J, Durrin LK, Wilkinson TA, Krontiris TG, Chen Y (2004) Identification of a SUMO-binding motif that recognizes SUMO-modified proteins. Proc Natl Acad Sci 101:14373–14378
Hecker CM, Rabiller M, Haglund K, Bayer P, Dikic I (2006) Specification of SUMO1- and SUMO2-interacting motifs. J Biol Chem 281:16117–16127
Aguilar-Martinez E, Chen X, Webber A, Mould AP, Seifert A, Hay RT, Sharrocks AD (2015) Screen for multi-SUMO-binding proteins reveals a multi-SIM-binding mechanism for recruitment of the transcriptional regulator ZMYM2 to chromatin. Proc Natl Acad Sci 112:E4854–E4863
Sun H, Hunter T (2012) Poly-small ubiquitin-like modifier (PolySUMO)-binding proteins identified through a string search. J Biol Chem 287:42071–42083
Tatham MH, Geoffroy MC, Shen L, Plechanovova A, Hattersley N, Jaffray EG, Palvimo JJ, Hay RT (2008) RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation. Nat Cell Biol 10:538–546
Rodriguez MS, Dargemont C, Hay RT (2001) SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting. J Biol Chem 276:12654–12659
Hendriks IA, D’Souza RC, Yang B, Verlaan-de Vries M, Mann M, Vertegaal AC (2014) Uncovering global SUMOylation signaling networks in a site-specific manner. Nat Struct Mol Biol 21:927–936
Psakhye I, Jentsch S (2012) Protein group modification and synergy in the SUMO pathway as exemplified in DNA repair. Cell 151:807–820
Ulrich HD (2008) The fast-growing business of SUMO chains. Mol Cell 32:301–305
Sriramachandran AM, Dohmen RJ (2014) SUMO-targeted ubiquitin ligases. Biochim Biophys Acta 1843:75–85
Guo Y, Kammerer RA, Engel J (2000) The unusually stable coiled-coil domain of COMP exhibits cold and heat denaturation in 4-6 M guanidinium chloride. Biophys Chem 85:179–186
Chichili V, Kumar V, Sivaraman J (2013) Linkers in the structural biology of protein-protein interactions. Protein Sci 22:153–167
Acknowledgements
This work was supported by the Wellcome Trust and a Royal Society-Wolfson award to (A.D.S.)
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Aguilar-Martínez, E., Sharrocks, A.D. (2016). The Use of Multimeric Protein Scaffolds for Identifying Multi-SUMO Binding Proteins. 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_14
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DOI: https://doi.org/10.1007/978-1-4939-6358-4_14
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