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KMID : 0545120040140051022
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Journal of Microbiology and Biotechnology
2004 Volume.14 No. 5 p.1022 ~ p.1030
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Cloning, Expression, and Characterization of DNA Polymerase from Hyperthermophilic Bacterium Aquifex pyrophilus
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Choi JJ
Kwon ST
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Abstract
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The gene encoding Aquifex pyrophilus (Apy) DNA polymerase was cloned and sequenced. The Apy DNA polymerase gene consists of 1725 bp coding for a protein with 574 amino acid residues. The deduced amino acid sequence of Apy DNA polymerase showed a high sequence homology to Escherichia coli DNA polymerase I-like DNA polymerases. It was deduced by amino acid sequence alignment that Apy DNA polymerase like the Klenow fragment has only the two domains the 3→5 exonuclease domain and the 5→3 polymerase domain containing the characteristic motifs. The Apy DNA polymerase gene was expressed under the control of T7lac promoter on the expression vector pET-22b(+) in E. coli. The expressed enzyme was purified by heat treatment and Cibacron blue 3GA and UNOTM Q column chromatographies. The optimum pH of the purified enzyme was 7.5 and the optimal concentrations of KCl and Mg2+ were 20 mM and 3 mM respectively. Apy DNA polymerase contained a double stranddependent 3→5 proofreading exonuclease activity but lacked any detectable 5→3 exonuclease activity which is consistent with its amino acid sequence. The somewhat lower thermostability of Apy DNA polymerase than the growth temperature of A. pyrophilus was analyzed by the comparison of amino acid composition and pressure effect.
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