title: Production and Purification of Recombinant SUMOylated Proteins Using Engineered Bacteria.
abstract: SUMO is a ubiquitin-like protein that is covalently conjugated to numerous cellular proteins to modify their function and fate. Although large progresses have been made in the identification of SUMOylated proteins, the molecular consequences of their SUMOylation are generally unknown. This is, most often, due to the low abundance of SUMOylated proteins in the cell, usually less than 1 % of a given protein being modified at steady state. To gain insights into the role of specific SUMOylation targets, SUMO conjugation can be reconstituted in vitro using purified proteins. However, for most substrates, the efficiency of in vitro SUMOylation is too low to obtain sufficient amounts of their SUMOylated forms for biochemical studies. Here, we describe a detailed protocol to purify large amounts of recombinant SUMOylated proteins using bacteria modified to express His-tagged SUMO as well as the SUMO-activating and -conjugating enzymes.
authors.affiliation: Equipe Labellisée par la Ligue contre le Cancer, Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535, Université de Montpellier, 1919 Route de Mende, 34293, Montpellier cedex 5, France.
authors.forename: Frédérique
authors.initials: F
authors.lastname: Brockly
authors.affiliation: Equipe Labellisée par la Ligue contre le Cancer, Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535, Université de Montpellier, 1919 Route de Mende, 34293, Montpellier cedex 5, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Piechaczyk
authors.affiliation: Equipe Labellisée par la Ligue contre le Cancer, Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535, Université de Montpellier, 1919 Route de Mende, 34293, Montpellier cedex 5, France. guillaume.bossis@igmm.cnrs.fr.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Bossis
mesh_terms: D001671:Biochemistry / Q000379:methods*; D060846:Cell Engineering / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D037601:Mannose-Binding Lectin / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D058207:Sumoylation; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
chemical_list: C471213:His-His-His-His-His-His; D037601:Mannose-Binding Lectin; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; C492189:SUMO3 protein, human; D014452:Ubiquitins; D006639:Histidine; D048031:JNK Mitogen-Activated Protein Kinases
keywords: Biochemistry; Protein purification; SUMOylation