Affinity Chromatography of DNA‐Binding Enzymes on Single‐Stranded DNA‐Agarose Columns

Abstract

Agarose gels containing immobilized single‐stranded circular DNA from phage fd or denatured calf thymus DNA were investigated for their use in the affinity chromatography of DNA‐binding enzymes. The DNA content of gel fragments is stable under the conventional conditions of enzyme purification. Single‐stranded DNA‐agarose columns have a high capacity to bind DNA‐specific proteins. They were used to differentiate between similar enzymatic activities in DNA‐free extracts from Escherichia coli. Preparative purification is described for the following enzymes: E. coli DNA polymerase I, DNA polymerase II, RNA polymerase, exonuclease III and T4 polynucleotide kinase. Enzyme purification was as high as 200‐fold, recovery of enzymatic activity was 75–100%. Copyright © 1972, Wiley Blackwell. All rights reserved