Thermostable DNA polymerase from the archaebacterium Sulfolobus acidocaldarius. Purification, characterization and immunological properties

Eur J Biochem. 1989 Jan 2;178(3):619-26. doi: 10.1111/j.1432-1033.1989.tb14490.x.

Abstract

We have purified to near homogeneity a DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a polypeptide of 100 kDa. On the basis of a Stokes radius of 4.2 nm and a sedimentation coefficient of 6 S, the purified enzyme has an estimated molecular mass of 109 kDa. These results are consistent with the enzyme being a monomer of 100 kDa. In addition a polyclonal antiserum, obtained by injection of the electroeluted 100-kDa polypeptide into a rabbit, specifically neutralized the DNA-polymerase activity. The enzyme is sensitive to both N-ethylmaleimide and 2',3'-dideoxyribosylthymine triphosphate and resistant to aphidicolin. The purified DNA polymerase has neither exonuclease nor primase activities. In our in vitro conditions, the enzyme is thermostable up to 80 degrees C and is active between 55 degrees C and 85 degrees C in the presence of activated calf-thymus DNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Archaea / enzymology*
  • Bacteria / enzymology*
  • Centrifugation, Density Gradient / methods
  • Chromatography, Affinity / methods
  • Chromatography, Ion Exchange / methods
  • DNA-Directed DNA Polymerase / isolation & purification*
  • DNA-Directed DNA Polymerase / metabolism
  • Enzyme Stability
  • Hot Temperature
  • Indicators and Reagents
  • Kinetics
  • Macromolecular Substances
  • Molecular Weight
  • Templates, Genetic

Substances

  • Indicators and Reagents
  • Macromolecular Substances
  • DNA-Directed DNA Polymerase