title: The Use of Multimeric Protein Scaffolds for Identifying Multi-SUMO Binding Proteins.
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.
authors.affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Manchester, M13 9PT, UK. elisa.aguilar-martinez@manchester.ac.uk.
authors.forename: Elisa
authors.initials: E
authors.lastname: Aguilar-Martínez
authors.affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Manchester, M13 9PT, UK.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Sharrocks
mesh_terms: D064236:Cartilage Oligomeric Matrix Protein / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D058207:Sumoylation; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
chemical_list: D064236:Cartilage Oligomeric Matrix Protein; D005982:Glutathione Transferase; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014452:Ubiquitins; C492189:SUMO3 protein, human; C577553:TSP5 protein, human
keywords: Multi-SUMO; Poly-SUMO; Protein purification; Protein–protein interaction; Pull-down