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  • Title: Improved method for preparation of ubiquitin-ligated lysozyme as substrate of ATP-dependent proteolysis.
    Author: Tamura T, Tanaka K, Tanahashi N, Ichihara A.
    Journal: FEBS Lett; 1991 Nov 04; 292(1-2):154-8. PubMed ID: 1659994.
    Abstract:
    A simple method was developed for preparation of proteins conjugated with ubiquitin. Heat-denatured 125I-labeled lysozyme was highly ubiquitinated by incubation at pH 9.0 with a ubiquitin-protein ligase system consisting of E1, E2 and E3 that had been partially purified from rabbit reticulocytes by affinity chromatography with ubiquitin as a ligand. The resulting conjugates were separated from free lysozyme and other proteins by successive chromatographies on anion and cation ion-exchange resins. The ubiquitinated 125I-lysozymes recovered in the fraction not adsorbed to either resin served as an efficient substrate for ATP-dependent proteolysis in a reticulocyte lysate or with a purified 26 S protease complex. By the present method, 125I-lysozyme-Ub conjugates can be prepared in 3 h with a high yield of 15-20%.
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