Purification method of the COP9 signalosome from human erythrocytes.
The COP9 signalosome (CSN) is a multimeric protein complex that occurs in all eukaryotic cells. Originally described in plants as a regulator of photomorphogenesis, its purification and characterization from mammalian cells revealed significant sequence homologies to subunits of the 26S proteasome lid complex, as well as of the eukaryotic translation initiation factor 3. Recent studies disclosed its participation in processes such as DNA repair, cell cycle regulation, development, and angiogenesis. At the moment, the pleiotropic effects of the CSN point to a regulatory role in the ubiquitin/26S proteasome system, but its exact function still remains to be clarified. This chapter describes the method to purify human CSN from red blood cells. Two outdated erythrocyte concentrates are sufficient to prepare approximately 0.5 mg of CSN. Washed cells are first lysed and then proteins are separated by a DEAE anion-exchange column. The CSN-containing fractions are pooled and subjected to an ammonium sulfate precipitation followed by dialysis. The concentrated proteins are then loaded onto a glycerol density gradient and ultracentrifugation is performed. The purification procedure is continued using two succeeding anion-exchange columns, resulting in a sufficiently pure CSN complex. Optionally, an additional density gradient centrifugation can be attached. The purified CSN complex possesses kinase, deneddylase, and deubiquitinase activities and can be stored for at least 2 months on ice at 4 degrees .
Hetfeld
Bettina K J
BK
Department of Surgery, Division of Molecular Biology, Medical Faculty Charité, Humboldt University, 10117 Berlin, Germany.
Bech-Otschir
Dawadschargal
D
Dubiel
Wolfgang
W
D015153:Blotting, Western; D000075686:COP9 Signalosome Complex; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D046912:Multiprotein Complexes / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*; D010447:Peptide Hydrolases / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*; D012394:Rosaniline Dyes
D046912:Multiprotein Complexes; D012394:Rosaniline Dyes; C048139:Coomassie blue; D010447:Peptide Hydrolases; D000075686:COP9 Signalosome Complex