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.
Copyright 1999 Academic Press.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Triphosphatases / genetics
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Adenosine Triphosphatases / isolation & purification*
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Amino Acid Motifs
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Animals
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Bloom Syndrome
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Chelating Agents
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Chromatography, Affinity / methods
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Chromatography, Gel / methods
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DNA Helicases / genetics
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DNA Helicases / isolation & purification*
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Edetic Acid / pharmacology
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Escherichia coli / genetics
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Gene Expression
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Histidine / metabolism*
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Humans
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Molecular Probes
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Nickel
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Nitrilotriacetic Acid / analogs & derivatives
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Nitrilotriacetic Acid / pharmacology
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Oligopeptides / drug effects
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Oligopeptides / isolation & purification*
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Organometallic Compounds / pharmacology
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RecQ Helicases
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Solubility
Substances
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Chelating Agents
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Molecular Probes
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Oligopeptides
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Organometallic Compounds
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nickel nitrilotriacetic acid
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Histidine
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Nickel
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Edetic Acid
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Adenosine Triphosphatases
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Bloom syndrome protein
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DNA Helicases
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RecQ Helicases
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Nitrilotriacetic Acid