Characterization of bacteriophage T7 DNA polymerase purified to homogeneity by antithioredoxin immunoadsorbent chromatography

J Biol Chem. 1981 Mar 25;256(6):3112-7.


DNA polymerase of bacteriophage T7 is composed of two subunits, the gene 5 protein of the phage and the host-coded thioredoxin. We have purified T7 DNA polymerase to homogeneity from T7-infected Escherichia coli B cells with a novel technique based on immunoadsorbent affinity chromatography. The enzyme binds quantitatively to a column of anti-thioredoxin Sepharose 4B and remains as an active complex in the immobilized state. It is eluted in fully active and highly purified form by a pulse of buffer at pH 12. After a final phosphocellulose chromatography, T7 DNA polymerase of better than 99% purity, as estimated from sodium dodecyl sulfate polyacrylamide gel electrophoresis, is obtained. Determination of the molecular weight by sedimentation equilibrium centrifugation gives a value of 112,000. Denaturing gels showed that the enzyme is composed of gene 5 protein (Mr = 87,000 +/- 3,000) and thioredoxin (Mr = 12,000) in a 1:1 stoichiometry. The amino acid composition of the enzyme and its spectrum was determined. The DNA polymerase activity is dependent on sulfhydryl compounds, sensitive to salt, and shows a comparatively high Km value for the four deoxyribonucleotides. The enzyme preparation has an inherent 3' leads to 5' exonuclease activity, attacking both native and denatured T7 DNA; it is free from detectable endonuclease activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / analysis
  • Chromatography, Affinity
  • DNA-Directed DNA Polymerase / isolation & purification*
  • DNA-Directed DNA Polymerase / metabolism
  • Drug Stability
  • Escherichia coli / enzymology*
  • Ethylmaleimide / pharmacology
  • Molecular Weight
  • Spectrophotometry, Ultraviolet
  • T-Phages / enzymology*
  • Thioredoxins / analysis


  • Amino Acids
  • Thioredoxins
  • DNA-Directed DNA Polymerase
  • Ethylmaleimide