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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Cloning, Expression, and Characterization of Thermostable DNA Polymerase from Thermoanaerobacter yonseiensis

  • Kim, Dae-Jin (Programs in Biomaterials Science and Engineering, College of Science, Yonsei University) ;
  • Jang, Hyeung-Jin (Programs in Biomaterials Science and Engineering, College of Science, Yonsei University) ;
  • Pyun, Yu-Ryang (Programs in Biomaterials Science and Engineering, College of Science, Yonsei University) ;
  • Kim, Yu-Sam (Department of Biochemistry, College of Science, Yonsei University)
  • Published : 2002.05.31
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Abstract

A gene, coined tay, for a thermostable DNA polymerase from the novel, extremely thermophilic bacterium Thermoanaerobacter yonseiensis was cloned and expressed in E. coli. Using a DNA polymerase homologous PCR product as a hybridization probe, tay was isolated and sequenced to consist of 2621 nucleotides that encode 872 amino acids. A database analysis showed that DNA polymerase, coined Tay, from T. yonseiensis shared a 39% to 47% identity in the amino acid sequence with those from other DNA polymerases. Tay was overexpressed in E. coli as a fusion protein with a poly-histidine tag at the C-terminus. It was purified by heat treatment, followed by a $Ni^{2+}$-chelate column. The molecular weight of purified Tay was approximately 97 kDa, as shown by SDS PAGE, and it showed high DNA polymerase activity and thermostability. However, it had no 3'$\rightarrow$5' exonuclease activity.

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References

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