Utility of polyhistidine-tagged ubiquitin in the purification of ubiquitin-protein conjugates and as an affinity ligand for the purification of ubiquitin-specific hydrolases.

摘要: The purification and biochemical characterization of protein substrates the ubiquitin-dependent pathway proteolysis is made difficult in part by low steady state levels ubiquitin-protein conjugates. We report here on use a polyhistidine-tagged ubiquitin molecule (HisUb) for conjugates metal chelate chromatography. When Escherichia coli extracts containing expressed HisUb were passed through nitrilotriacetic acid-agarose column immobilized Ni2+ ions (Ni-NTA column), was retained. After washing to remove unbound nonspecifically bound proteins, pH 4.5 wash used elute highly purified HisUb. Purified wild-type tested their ability form Ni(2+)-binding wheat germ vitro conjugation reaction. In some experiments, preincubated with iodoacetamide inhibit activating conjugating enzymes. Only those assays an ATP-regenerating system not pretreated produced significant multiple also examined potential as affinity ligand higher plant ubiquitin-specific hydrolases. As test, crude lysate E. expressing yeast hydrolase (Yuh1) Ni-NTA Yuh1 retained specifically eluted when equilibrated buffer ubiquitin.