IMPROVED METHOD FOR PREPARATION OF UBIQUITIN-LIGATED LYSOZYME AS SUBSTRATE OF ATP-DEPENDENT PROTEOLYSIS

A simple method was developed for preparation of proteins conjugated with ubiquitin. Heat-denatured I-125-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 I-125-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, I-125-lysozyme-Ub conjugates can be prepared in 3 h with a high yield of 15-20%.