title: Using Biotinylated SUMO-Traps to Analyze SUMOylated Proteins.
abstract: SUMO-interacting motifs (SIMs) recognize SUMOylated proteins with high specificity allowing to connect SUMO-modified proteins. Multiple SIMs fused to distinct tags have been used to increase their affinity and generate more efficient purification tools. Enrichment of SUMOylated proteins using SIMs arranged in tandem (SUMO-traps) facilitates the identification and characterization of protein targets in vitro and in vivo. Here a protocol to produce biotinylated SUMO-traps (bioSUBEs) to capture SUMO chains and typical SUMOylated proteins such as p53 or IkBα is presented. Biotinylated SUMO-traps represent an alternative to reduce the background associated to bigger tags, e.g., during mass spectrometry analysis. Consequently, bioSUBEs are alternative tools to characterize endogenous SUMO targets.
authors.affiliation: Ubiquitylation and Cancer MolecularBiology (UMCB) Laboratory, Inbiomed, 20009, San Sebastian-Donostia, Gipuzkoa, Spain.
authors.forename: Valérie
authors.initials: V
authors.lastname: Lang
authors.affiliation: Ubiquitylation and Cancer MolecularBiology (UMCB) Laboratory, Inbiomed, 20009, San Sebastian-Donostia, Gipuzkoa, Spain.|Group of Ubiquitin-Dependent Proteolysis and Intracellular Communication (G(u)ic), Institute of Biomedical Imaging and Life Sciences (IBILI), University of Coimbra, 3000-548, Coimbra, Portugal.
authors.forename: Elisa
authors.initials: E
authors.lastname: Da Silva-Ferrada
authors.affiliation: CIC bioGUNE, Bizkaia Technology Park, 48160, Derio, Bizkaia, Spain.
authors.forename: Rosa
authors.initials: R
authors.lastname: Barrio
authors.affiliation: CIC bioGUNE, Bizkaia Technology Park, 48160, Derio, Bizkaia, Spain.
authors.forename: James D
authors.initials: JD
authors.lastname: Sutherland
authors.affiliation: INBIOMED, San Sebastian, Spain. manuel.rodriguez@itav.fr.
authors.forename: Manuel S
authors.initials: MS
authors.lastname: Rodriguez
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000072000:NF-KappaB Inhibitor alpha / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D058207:Sumoylation; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000906:Antibodies; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C117909:SUMO2 protein, human; C492189:SUMO3 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D016159:Tumor Suppressor Protein p53; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha; D005982:Glutathione Transferase; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli
keywords: Analysis; Biotinylation; BirA; Purification; SIMs; SUBEs; SUMOylation