Purification and properties of Thermus filiformis DNA polymerase expressed in Escherichia coli.
The gene encoding Thermus filiformis (Tfi) DNA polymerase was expressed under the control of the tac promoter on a high-copy plasmid, pJR, in Escherichia coli. The Tfi DNA polymerase was purified by using heat treatment and DEAE-Sephacel column chromatography. The purified enzyme had a molecular mass of 92 kDa, as estimated by SDS/PAGE. The optimum pH and temperature of the enzyme were 8.4-9.0 and 70-72.5 degrees C respectively. The half-life of the enzyme at 94 degrees C was approx. 40 min. The enzyme was activated by the bivalent cations, Mg(2+) and Mn(2+), and was inhibited by EDTA. The optimal Mg(2+) concentration of the enzyme was 4 mM. The optimal conditions for the PCR reaction were slightly different from those for the enzyme activity except for the optimal Mg(2+) concentration. Low concentrations of KCl had no effect on either the enzymic activity or the PCR amplification. The result of the PCR experiment with the enzyme indicates that Tfi DNA polymerase might be useful in DNA amplification.
Choi
J J
JJ
Department of Genetic Engineering, Sungkyunkwan University, 300 Chunchon-Dong, Jangan-Ku, Suwon 440-746, Korea.
Jung
S E
SE
Kim
H K
HK
Kwon
S T
ST
D001483:Base Sequence; D002412:Cations; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D013696:Temperature; D013824:Thermus / Q000201:enzymology*
D002412:Cations; D017931:DNA Primers; D007202:Indicators and Reagents; D004259:DNA-Directed DNA Polymerase