Purification of overexpressed hexahistidine-tagged BLM N431 as oligomeric complexes.
BLM is a DNA helicase encoded by a gene which is mutated in persons with Bloom's syndrome. The protein is a member of the RecQ subfamily of helicases and contains a central domain constituted by the seven motifs conserved in all helicases. In contrast, the N-terminal portion of BLM lacks similarity to any other known proteins or motifs. We have expressed the first 431 amino acids of this domain as a fusion to a hexahistidine tag (BLM N431) in Escherichia coli. A method of purification was developed which involves elution from Ni-NTA resin in imidazole and EDTA, followed by treatment with DTT and gel filtration on Sephacryl-300. The treatment with EDTA and DTT prevents and disrupts aggregation of BLM N431. The purified protein appears to form hexamers and dodecamers, suggesting that the N-terminal domain of BLM is involved in the organization of the quaternary structure of BLM.
Beresten
S F
SF
Department of Human Genetics, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York, 10021, USA.
Stan
R
R
van Brabant
A J
AJ
Ye
T
T
Naureckiene
S
S
Ellis
N A
NA
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification*; D020816:Amino Acid Motifs; D000818:Animals; D001816:Bloom Syndrome; D002614:Chelating Agents; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D004265:DNA Helicases / Q000235:genetics / Q000302:isolation & purification*; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D015335:Molecular Probes; D009532:Nickel; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D009842:Oligopeptides / Q000187:drug effects / Q000302:isolation & purification*; D009942:Organometallic Compounds / Q000494:pharmacology; D053484:RecQ Helicases; D012995:Solubility
D002614:Chelating Agents; D015335:Molecular Probes; D009842:Oligopeptides; D009942:Organometallic Compounds; C088321:nickel nitrilotriacetic acid; D006639:Histidine; D009532:Nickel; D004492:Edetic Acid; D000251:Adenosine Triphosphatases; C097151:Bloom syndrome protein; D004265:DNA Helicases; D053484:RecQ Helicases; D009571:Nitrilotriacetic Acid