ABSTRACT
Poly(N-isopropylacrylamide)s with imidazole endgroups were used to separate a histidine-tagged protein fragment directly from a crude cell lysate. The polymers display a lower critical solution temperature that can be tuned to occur at a range of subambient temperatures. UV-visible spectra indicated differences in the binding in aqueous media of Cu(II) and Ni(II) to the imidazole endgroups. These changes in the UV-visible spectra were reflected in the solution/aggregation behavior of the polymers as studied by dynamic light scattering. The addition of Cu(II) disaggregated the polymers, and the polymer coil swelled. On the other hand, when Ni(II) was added the polymers remained aggregated in aqueous media. The polymers were used to purify residues 230-534 of the histidine-tagged breast cancer susceptibility protein his6-BRCA1. Cu(II) was found to be better suited to the formation of useful polymer-metal ion-protein complexes that display cloud points, since Ni(II)/polymer mixtures generated very little purified protein. The polymers were synthesized using a previously reported variation of the reversible addition-fragmentation chain termination (RAFT) methodology, using the chain transfer agent 3H-imidazole-4-carbodithioic acid 4-vinyl benzyl ester with N-isopropylacrylamide (NIPAM).
Subject(s)
Acrylic Resins/chemistry , BRCA1 Protein/chemistry , BRCA1 Protein/isolation & purification , Organometallic Compounds/chemistry , Acrylic Resins/chemical synthesis , Binding Sites , Copper/chemistry , Female , Histidine/chemistry , Humans , Molecular Structure , Nickel/chemistry , Organometallic Compounds/chemical synthesis , Osmolar Concentration , Scattering, Radiation , Spectrophotometry, Ultraviolet/methodsABSTRACT
The breast cancer susceptibility gene, BRCA1, encodes a large nuclear phosphoprotein, the major isoform of which is 1863 amino acids in size. Structure-function studies have been largely restricted to the only two domains identified by homology searches: the RING (really interesting new gene) and BRCT (BRCA1 C-terminus) domains. However, we have recently reported the identification of a large central soluble region of BRCA1 (residues 230-534) that binds specifically to four-way junction DNA, a property that potentially facilitates its role in the repair of DNA lesions by homologous recombination. We have now used a combination of limited proteolysis and extension cloning to identify more accurately the DNA-binding region of BRCA1. Limited trypsinolysis of BRCA1-(230-534) resulted in the production of a soluble domain identified as residues 230-339. However, after cloning, expression and purification of this region, studies revealed that it was unable to bind to four-way junctions, suggesting that the DNA-binding activity, in part, resides within residues 340-534. A series of fragments extending from residue 340 were produced, and each was tested for its ability to bind to four-way junction DNA in gel retardation assays. In these experiments, residues 340-554 of BRCA1 were identified as the minimal DNA-binding region. We then went on to characterize the conformation of this region using CD spectroscopy and analytical centrifugation.
Subject(s)
BRCA1 Protein/chemistry , BRCA1 Protein/metabolism , Breast Neoplasms/metabolism , DNA/metabolism , Amino Acid Sequence , BRCA1 Protein/genetics , BRCA1 Protein/isolation & purification , Binding Sites , Circular Dichroism , Cloning, Molecular , DNA/genetics , Disease Susceptibility , Gene Expression , Molecular Sequence Data , Protein Conformation , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Highly branched poly(NIPAM) have been prepared using the technique of reversible addition-fragmentation chain transfer (RAFT) polymerisation using a chain transfer agent that allows the incorporation of imidazole functionality in the polymer chain-ends. The lower critical solution temperature (LCST) of the polymers can be controlled by the amount of hydrophobe and GMA incorporated during copolymerisation procedures. These thermally responsive "smart" polymers were used to purify a His-tagged BRCA-1 protein fragment by affinity precipitation. [Diagram: see text]
Subject(s)
Acrylic Resins/metabolism , Methacrylates/metabolism , Polymers/metabolism , Propylene Glycol/metabolism , Proteins/metabolism , Polymers/chemical synthesis , Protein Binding/physiologyABSTRACT
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.
