Characterization and PCR applications of dUTPase from the hyperthermophilic euryarchaeon Thermococcus pacificus |
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| Institution: | 1. Department of Biotechnology and Bioengineering, Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon 440-746, Republic of Korea;2. Rexgene Biotech Co., Ltd., 9 F Wooyoung Venture Bldg. 1330-13 Seocho-dong, Seocho-gu, Seoul 137-070, Republic of Korea;1. Department of Orthopedics, Urmia University of Medical Sciences, Orumiyeh, Iran;2. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA;3. Division of Hematology, Mayo Clinic, Rochester, MN;1. Department of Urology, Kingston Hospital, Galsworthy Road, Kingston upon Thames, Surrey KT2 7QB, UK;2. Division of Surgery and Interventional Science, University College London, 21 University Street, London WC1E 6AU, UK;3. Department of Urology, Whittington Hospitals NHS Trust, Magdala Avenue, London N19 5NF, UK;4. Division of Surgery, Department of Surgery and Cancer, Imperial College London, South Kensington Campus, London SW7 2AZ, UK;5. Imperial Urology, Imperial College Healthcare NHS Trust, Fulham Palace Road, London W6 8RF, UK;1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, PR China;2. Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai, 200237, PR China;1. Research Center for Tropical and Infectious Disease, Kerman University of Medical Sciences, Kerman, Iran;2. Department of Medical Microbiology, Kerman University of Medical Sciences, Kerman, Iran;3. Department of Medical Microbiology, Kerman University of Medical Sciences, Kerman, Iran;3. From the Genome and Systems Biology Degree Program, National Taiwan University and Academia Sinica, Taipei 10617,;4. the Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, and;5. the Center for Systems Biology, National Taiwan University, Taipei 10617, Taiwan |
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| Abstract: | We cloned and sequenced the gene encoding Thermococcus pacificus dUTPase (Tpa dUTPase). The Tpa dUTPase gene consists of 471 bp and encodes a 156-amino acid protein. The deduced amino acid sequence of Tpa dUTPase has high sequence similarity with other archaeal dUTPases. The Tpa dUTPase had an 18-kDa major protein band consistent with the 17,801 Da molecular mass calculated based on the amino acid sequence. The specific activity of Tpa dUTPase on dUTP at 85 °C was 90,909 U/mg. For Tpa dUTPase activity, we determined an optimum pH of 8.5 and temperature of 85 °C. Magnesium ions strongly induced activity, with an optimum concentration of 0.75 mM. The half-life of the enzyme at 94 °C was about 7 h. The specific activity of the Tpa dUTPase on dUTP was about 10–20-fold higher than that of Tpa dUTPase on dCTP. Tpa dUTPase enhanced the PCR amplification efficiency of long targets when Pfu and Vent DNA polymerases were used. |
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