Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Purification and Characterization of the N-terminally Truncated DNA Polymerase from Thermus thermophilus HJ6

Thermus thermophilus HJ6 유래 N-말단 결실 DNA Polymerase의 정제 및 특성

  • Jeon, Sung-Jong (Department of Biotechnology & Bioengineering, Dong-Eui University) ;
  • Seo, Min-Ho (Department of Biotechnology & Bioengineering, Dong-Eui University)
  • Received : 2010.03.04
  • Accepted : 2010.03.31
  • Published : 2010.06.28
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Abstract

The gene encoding N-terminally truncated Tod polymerase ($\Delta$Tod polymerase) from Thermus thermophilus HJ6 was expressed in Escherichia coli under the control of the lambda pR and pL tandem promoters on the expression vector pJLA503. The N-terminal domain (250 amino acids) of Tod polymerase was removed without significant effect on enzyme activity and stability, while no 5'$\rightarrow$3' exonuclease activity was detected. The $\Delta$Tod polymerase was verified to possess very efficient reverse transcriptase (RT) activity in the presence of $MgCl_2$. The cDNA can also be amplified in the polymerase chain reaction (PCR) with this mutant enzyme. The $\Delta$Tod polymerase was exhibited higher activity than the Taq polymerase in a one-step RT-PCR.

고온균 Thermus thermophilus HJ6 유래의 N-말단 결실 Tod polymerase($\Delta$Tod polymerase)는 온도 감수성 프로모터 (lambda pR and pL)를 포함하는 pJLA503 벡터를 이용하여 대장균에서 발현하였다. N-말단 250개 아미노산이 제거된 $\Delta$Tod polymerase는 5'$\rightarrow$3' exonuclease 활성은 없어지고 DNA 중합반응의 활성은 그대로 유지되었다. $\Delta$Tod polymerase는 $MgCl_2$의 존재 하에서 매우 효율적으로 역전사 반응과 PCR 반응을 수행하였다. 또한 $\Delta$Tod polymerase는 one-step RT-PCR 반응에서 Taq polymerase 보다 높은 cDNA 증폭 효율을 나타내었다.

Keywords

References

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