Purification, crystallization and preliminary X-ray crystallographic analysis of Pyrococcus furiosus DNA polymerase

Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):986-8. doi: 10.1107/s0907444998000353.

Abstract

DNA polymerase gene from the hyperthermophilic Archaeon Pyrococcus furiosus has been cloned and the protein overexpressed in Escherichia coli to produce an active enzyme. The purified protein was crystallized from 0.08 M ammonium sulfate, 0.05 M Na-cacodylate, pH 6.5, 0.15%(v/v) NP40, 0.05%(v/v) Tween 20 and 4.5%(w/v) polyethylene glycol 6000 by the vapour-diffusion method. The orthorhombic crystals had unit-cell dimensions of a = 92.5, b = 125.4, c = 192.1 A; alpha = beta = gamma = 90 degrees. The crystals diffracted beyond 4 A on a 1.08 A synchrotron radiation source.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Cloning, Molecular
  • Crystallization
  • Crystallography, X-Ray
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / isolation & purification
  • Protein Conformation*
  • Pyrococcus furiosus / enzymology*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification

Substances

  • Recombinant Fusion Proteins
  • Pfu DNA polymerase
  • DNA-Directed DNA Polymerase