Improved method for preparation of ubiquitin‐ligated lysozyme as substrate of ATP‐dependent proteolysis

摘要

>A simple method was developed for preparation of proteins conjugated with ubiquitin. Heat‐denatured125I‐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 ubiquitinated125I‐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%.