Characterization of a family B DNA polymerase from the hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN4/I and its application to PCR.
A family B DNA polymerase gene from the hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN4/I was highly expressed under the control of T7lac promoter of pET-28ARG in Escherichia coli BL21-CodonPlus(DE3)-RIL cells. The produced I. hospitalis (Iho) DNA polymerase was purified by heat treatment followed by HisTrap™ HP column and HiTrap™ SP column chromatographies. The molecular mass of the purified Iho DNA polymerase was 88 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH for Iho DNA polymerase activity was 7.0 and the optimal temperature was 70 °C. Iho DNA polymerase was strongly activated by the presence of magnesium ion at an optimum concentration of 3 mM. The optimal concentration of KCl for Iho DNA polymerase activity was 60 mM. The half-life of the enzyme at 94 °C was about 2 h. The optimal conditions for polymerase chain reaction (PCR) were determined. Iho DNA polymerase possesses 3'→5' exonuclease activity, and the fidelity of the Iho DNA polymerase was similar to that of Pfu and Vent DNA polymerases. However, Iho DNA polymerase provided more enhanced efficiency of PCR amplification than Pfu and Vent DNA polymerases. Iho DNA polymerase could successfully amplify a 2-kb λ DNA target with a 10-s extension time and could amplify a DNA fragment up to 8 kb λ DNA.
Department of Biotechnology and Bioengineering, Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon, 440-746, South Korea.
Kang-Jin
KJ
Seo
Sung Suk
SS
Cho
Hye Woo
HW
Ppyun
Man-Hui
MH
Youn
Seung Hyun
SH
Kim
Bo-Sung
BS
Seo
Suk-Tae
ST
Kwon
D000595:Amino Acid Sequence; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019620:Desulfurococcaceae / Q000201:enzymology*; D005092:Exonucleases / Q000378:metabolism; D006358:Hot Temperature; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment
D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases