Utility of polyhistidine-tagged ubiquitin in the purification of ubiquitin-protein conjugates and as an affinity ligand for the purification of ubiquitin-specific hydrolases.
The purification and biochemical characterization of protein substrates of the ubiquitin-dependent pathway of proteolysis is made difficult in part by the low steady state levels of ubiquitin-protein conjugates. We report here on the use of a polyhistidine-tagged ubiquitin molecule (HisUb) for the purification of ubiquitin-protein conjugates by metal chelate chromatography. When Escherichia coli extracts containing expressed HisUb were passed through a nitrilotriacetic acid-agarose column containing immobilized Ni2+ ions (Ni-NTA column), HisUb was retained. After washing to remove unbound and nonspecifically bound proteins, a pH 4.5 wash was used to elute highly purified HisUb. Purified HisUb and wild-type ubiquitin were tested for their ability to form Ni(2+)-binding ubiquitin-protein conjugates in a wheat germ in vitro conjugation reaction. In some experiments, wheat germ extracts were preincubated with iodoacetamide to inhibit ubiquitin activating and conjugating enzymes. Only those conjugation assays containing HisUb and an ATP-regenerating system not pretreated with iodoacetamide produced significant levels of multiple Ni(2+)-binding ubiquitin-protein conjugates. We also examined the potential of HisUb as an affinity ligand for the purification of higher plant ubiquitin-specific hydrolases. As a test, a crude lysate of E. coli expressing a yeast ubiquitin-specific hydrolase (Yuh1) was passed through a Ni-NTA column containing bound HisUb. Yuh1 was retained on the column and was specifically eluted when the column was equilibrated with buffer containing wild-type ubiquitin.
Beers
E P
EP
Section of Molecular and Cellular Biology, University of California, Davis 95616.
Callis
J
J
D000595:Amino Acid Sequence; D002614:Chelating Agents; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005822:Genetic Vectors; D006639:Histidine; D006867:Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D008024:Ligands; D008670:Metals; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D013379:Substrate Specificity; D014908:Triticum / Q000737:chemistry; D014452:Ubiquitins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D002614:Chelating Agents; D008024:Ligands; D008670:Metals; D010455:Peptides; D010940:Plant Proteins; D014452:Ubiquitins; C033223:polyhistidine; D006639:Histidine; D006867:Hydrolases