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.
Keywords:
Multi-SUMO; Poly-SUMO; Protein purification; Protein–protein interaction; Pull-down.
MeSH terms
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Cartilage Oligomeric Matrix Protein / genetics
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Cartilage Oligomeric Matrix Protein / metabolism*
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Cell Line
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Cloning, Molecular
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Gene Expression
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Genetic Vectors / chemistry
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Genetic Vectors / metabolism
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Glutathione Transferase / genetics
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Glutathione Transferase / metabolism*
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Humans
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Protein Binding
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Protein Interaction Mapping
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Protein Processing, Post-Translational*
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Proteomics / methods*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / isolation & purification*
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Recombinant Fusion Proteins / metabolism
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Sumoylation
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Ubiquitins / genetics
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Ubiquitins / metabolism*
Substances
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COMP protein, human
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Cartilage Oligomeric Matrix Protein
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Recombinant Fusion Proteins
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Recombinant Proteins
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SUMO3 protein, human
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Ubiquitins
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Glutathione Transferase