Purification and Characterization of a Thermostable DNA Polymerase from the Hyperthermophilic Archaea Thermococcus fumicolans
Jean-Paul RAFFIN* and Jacques DIETRICH
Laboratoire de Biotechnologie des Micro-organismes Hydrothermaux, Ifremer, DRV/VP, B.P. 70, 29280 Plouzane, France
The archaea Thermococcus fumicolans is an obligatory chemo-organotrophic, strictly anaerobic and grows preferentially in the presence of elemental sulfur (1). As a parallel to the cloning, expression and characterization of the recombinant enzymatic form, the present study describes the purification of the native DNA polymerase from Thermococcus fumicolans. Heat stable DNA polymerases, such as Taq DNA polymerase, have been key elements in the development of the Polymerase Chain Reaction. Purification of the native enzyme included lysozyme treatment, followed by DNA precipitation by Polymin P. Purification of the enzyme included ammonium sulfate precipitation followed by chromatography on phosphocellulose, Phenyl sepharose, Source Q, Source S, Hi-Trap Heparin and Mono-Q. The process resulted in a more than 1200 fold purification and the purified enzyme, when stored at -20℃, in the presence of glycerol and Triton X100, was highly stable. The optimal temperature for activity was higher than 80℃ (limited by DNA stability) and the enzyme was rather halophilic (optimal K+ concentration higher than 200mM). The enzyme displayed a 3'-5' exonuclease proof-reading as well as a 5'-3' exonuclease activity. The molecular weight was of about 90 kDa.
1. Godfroy, A., Meunier, J.R., Guezennec, J., Lesongeur, F., Raguenes, G., Rimbault, A., and Barbier, G. (1996). Int. J. of Syst. Bacteriol., 46, 1113-1119.
