PURIFICATION OF OVEREXPRESSED HEXAHISTIDINE-TAGGED BLM N431 AS OLIGOMERIC COMPLEXES

Abstract 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.