An oligodeoxynucleotide affinity column for the isolation of sequence specific DNA binding proteins.
A nucleic acid affinity matrix containing a short oligodeoxynucleotide ligand has been prepared as an example of a material which can be used for the rapid and effective isolation of sequence specific DNA binding proteins. Two complementary oligodeoxynucleotides have been employed, one of which contains a small 5'-spacer arm with a terminal thiol group. Using this terminal thiol group, the ligand can be covalently coupled to Tresyl-activated Sepharose 4B or Epoxy-activated Sepharose 6B via a thioether linkage. This approach allows the specific attachment of the nucleic acid ligand via its 5'-terminus to the insoluble matrix. The double stranded affinity material was obtained by annealing of the complementary DNA fragment. As an example, we have used an eicosomer affinity column containing the sequence d(GAATTC) for the isolation of the Eco RI restriction endonuclease. Using a single column, the enzyme could be isolated by eluting the column with a single step or multistep gradient of increasing salt concentration. The enzyme was purified to 75%-85% homogeneity with yields of 0.1 mg to 0.2 mg from 0.5 g of cell paste.
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Department of Chemistry, Boston College, Chestnut Hill, MA 02167.
McLaughlin
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D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification*; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010957:Plasmids
D004268:DNA-Binding Proteins; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides