Premium
Improved method for preparation of ubiquitin‐ligated lysozyme as substrate of ATP‐dependent proteolysis
Author(s) -
Tomohiro Tamura,
Keiji Tanaka,
Nobuyuki Tanahashi,
Akira Ichihara
Publication year - 1991
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(91)80856-x
Subject(s) - lysozyme , proteolysis , ubiquitin , chemistry , biochemistry , lysis , ion chromatography , chromatography , substrate (aquarium) , reticulocyte , ubiquitin ligase , enzyme , yield (engineering) , trypsin , protease , rna , biology , ecology , materials science , metallurgy , gene
A simple method was developed for preparation of proteins conjugated with ubiquitin. Heat‐denatured 125 I‐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 125 I‐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, 125 I‐lysozyme‐Ub conjugates can be prepared in 3 h with a high yield of 15–20%.