Purification and characterization of Thermus caldophilus GK24 DNA polymerase.
A thermostable DNA polymerase from Thermus caldophilus GK24 was purified to near homogeneity by chromatographic methods, including ion-exchange, gel-filtration and affinity chromatography. The purified enzyme had a specific activity of 8400 U/mg at 75 degrees C and a molecular mass of 95 kDa, estimated by SDS/PAGE and Superose-12 gel filtration. Reaction conditions were investigated in terms of pH, metal-ion concentration and temperature. Experimental results showed that T. caldophilus (Tca) DNA polymerase had a maximum activity near pH 8.7 at 75 degrees C. The N-terminal sequence of the enzyme was highly similar to that of Thermus aquaticus (Taq) DNA polymerase, which was consistent with the fact that the enzyme had 5'-to-3' exonuclease activity and no 3'-to-5' exonuclease activity. Gene amplification using Tca DNA polymerase resulted in longer products than amplification using Taq DNA polymerase.
Molecular Biology Laboratory, Korea Institute of Science and Technology.
J H
JH
Park
J S
JS
Kim
S T
ST
Kwon
D S
DS
Lee
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D013824:Thermus / Q000201:enzymology*
D004259:DNA-Directed DNA Polymerase