Thermus thermophilus HJ6 유래 N-말단 결실 DNA Polymerase의 정제 및 특성
Purification and Characterization of the N-terminally Truncated DNA Polymerase from Thermus thermophilus HJ6.
0
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
고온균 Thermus thermophilus HJ6 유래의 N-말단 결실Tod polymerase(ΔTod polymerase)는 온도 감수성 프로모터(lambda pR and pL)를 포함하는 pJLA503 벡터를 이용하여대장균에서 발현하였다. N-말단 250개 아미노산이 제거된ΔTod polymerase는 5'→3' exonuclease 활성은 없어지고DNA 중합반응의 활성은 그대로 유지되었다. ΔTod polymerase는MgCl2 의 존재 하에서 매우 효율적으로 역전사 반응과 PCR 반응을 수행하였다. 또한 ΔTod polymerase는one-step RT-PCR 반응에서 Taq polymerase 보다 높은cDNA 증폭 효율을 나타내었다.
Abstract
The gene encoding Nterminally truncated Tod polymerase (ΔTod 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'→3' exonuclease activity was detected. The ΔTod polymerase was verified to possess very efficient reverse transcriptase (RT) activity in the presence of MgCl2. The cDNA can also be amplified in the polymerase chain reaction (PCR) with this mutant enzyme. The ΔTod polymerase was exhibited higher activity than the Taq polymerase in a one-step RT-PCR.
참고문헌 (13)
- Chien, A, "Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus", J. Bacteriol., 127, pp.1550~1557 (1976)
- Choi, J. J, "Purification and characterization of the 5'→3' exonuclease domaindeleted Thermus filiformis DNA polymerase expressed in Escherichia coli.Biotech.Lett.23:1647-1652", Biotech. Lett., 23, pp.1647~1652 (2001)
- Choi, J. J, "Purification and properties of Thermus filiformis DNA polymerase expressed in Escherichia coli. Biotechnol", Appl. Biochem., 30, pp.19~25 (1999)
- Frohman, M. A, "Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer", Proc. Natl. Acad. Sc.i USA., 85, pp.8998~9002 (1988)
- Gibbs, R. A, "Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of in vitro amplified cDNA", Proc Natl Acad Sci USA., 86, pp.1919~1923 (1989)
- Grebennikova, T. V, "Use of thermostable DNA polymerase from Thermus thermophilus KTP in a combined reverse transcription and amplification reaction of detecting interleukin 2alpha RNA and determining expression of the multidrug resistance gene (MDR-1)", Mol. Biol. (Mosk)., 29, pp.930~941 (1995)
- Kawasaki, E. S, "Diagnosis of chronic myeloid and acute lymphocytic leukemias by detection of leukemia-specific mRNA sequences amplified in vitro", Proc. Natl. Acad. Sc.i USA., 85, pp.5698~5702 (1988)
- Lawyer, F. C, "Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus", J. Bio.l Chem., 264, pp.6427~6437 (1989)
- Myers, T. M, "Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase", Biochemistry, 30, pp.7661~7666 (1991)
- 서민호, "Thermus thermophilus HJ6 유래 내열성 DNA Polymerase의 유전자 클로닝 및 발현", 한국미생물·생명공학회지, 37(1), pp.17~23 (2009), 한국미생물·생명공학회
- Sorokine, I, "Exonuclease activities associated with DNA polymerase α and β of the archaebacterium Halobacterium halobium", Eur. J. Biochem., 197, pp.781~784 (1991)
- Tse, W. T, "Reverse transcription and direct amplification of cellular RNA transcripts by Taq polymerase", Gene, 88, pp.293~296 (1990)
- Veres, G, "The molecular basis of the sparse fur mouse mutation", Science, 237, pp.415~417 (1987)