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 Bl oom'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 amin o acids of this domain as a fusion to a hexahistidine tag (BLM N431) in Esc herichia coli. A method of purification was developed which involves elutio n from Ni-NTA resin in imidazole and EDTA, followed by treatment with DTT a nd gel filtration on Sephacryl-300. The treatment with EDTA and DTT prevent s and disrupts aggregation of BLM N431. The purified protein appears to for m hexamers and dodecamers, suggesting that the N-terminal domain of BLM is involved in the organization of the quaternary structure of BLM. (C) 1999 A cademic Press.