Purification and properties of Aquifex aeolicus DNA polymerase expressed in Escherichia coli

FEMS Microbiol Lett. 2001 Jul 10;201(1):73-7. doi: 10.1111/j.1574-6968.2001.tb10735.x.

Abstract

The gene encoding Aquifex aeolicus (Aae) DNA polymerase was expressed under the control of the trp promoter on a high-copy plasmid, pTRPNS, in Escherichia coli. The expressed enzyme was purified 11-fold with a 13.8% yield and a specific activity of 2268.3 U mg(-1). The optimum pH of the enzyme was 6.8-7.2. The optimal concentrations of KCl and Mg(2+) were 20-30 mM and 4-5 mM, respectively. Aae DNA polymerase contained a double-strand-dependent 3'-->5' proofreading exonuclease activity but lacked any detectable 5'-->3' exonuclease activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cations, Divalent / pharmacology
  • Cloning, Molecular
  • DNA / metabolism
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / isolation & purification*
  • DNA-Directed DNA Polymerase / metabolism*
  • Edetic Acid / pharmacology
  • Escherichia coli / genetics*
  • Exonucleases / metabolism
  • Gram-Negative Aerobic Rods and Cocci / enzymology*
  • Gram-Negative Aerobic Rods and Cocci / genetics
  • Hydrogen-Ion Concentration
  • Magnesium / pharmacology
  • Potassium Chloride / pharmacology
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Temperature
  • Transformation, Bacterial

Substances

  • Cations, Divalent
  • Recombinant Proteins
  • Potassium Chloride
  • DNA
  • Edetic Acid
  • DNA-Directed DNA Polymerase
  • Exonucleases
  • Magnesium