Purification and characterisation of a soluble N-terminal fragment of the breast cancer susceptibility protein BRCA1.
The BRCA1 gene encodes a large multidomain protein of 1863 residues, mutations in which lead to breast cancer. Studies to elucidate the mechanisms by which BRCA1 prevents tumour formation have been restricted by the size of the protein. Unable to purify large amounts of the full-length protein, we have identified a fragment of BRCA1, amino acid residues 230-534, that when cloned into the expression vector pET 22b and expressed in Escherichia coli is found predominantly in the soluble portion of the cell lysate. The resulting protein was purified to homogeneity and studies reveal that BRCA1 230-534 binds specifically to four-way junction DNA when compared to duplex and single-stranded DNA.
Sturdy
Alice
A
Department of Chemistry, University of Sheffield, Dainton Building, Sheffield S3 7HF, UK.
Naseem
Riffat
R
Webb
Michelle
M
D019313:BRCA1 Protein / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics*; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D006801:Humans; D012995:Solubility; D020349:Surface Plasmon Resonance; D014461:Ultracentrifugation
D019313:BRCA1 Protein; D017931:DNA Primers; D004247:DNA