Characterization and PCR application of a new high-fidelity DNA polymerase from Thermococcus waiotapuensis |
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| Institution: | 1. Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland;2. Institute of Chemistry and Bioanalytics, University of Applied Sciences Northwestern Switzerland, Hofackerstrasses 30, 4132 Muttenz, Switzerland;1. Department of Forensic Medicine, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China;2. Department of Pathology, Fudan University Shanghai Cancer Centre, Shanghai, 200032, China;3. Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China;4. Key Laboratory of Glycoconjugate Research, Ministry of Health, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China;3. From New England Biolabs, Inc., Ipswich, Massachusetts 01938 and;4. the National Institute of Standards and Technology, Rockville, Maryland 20850;1. Department of Microbiology, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland;2. Collection of Plasmids and Microorganisms, University of Gdansk, Wita Stwosza 59, 80-308 Gdansk, Poland;3. Matis ohf, Vinlandsleid 12, Reykjavik 113, Iceland;4. Faculty of Life and Environmental Sciences, University of Iceland, Sæmundargötu 2, Reykjavik 101, Iceland;5. Prokazyme ehf, Vinlandsleid 14, Reykjavik 113, Iceland;6. A&A Biotechnology, Aleja Zwyciestwa 96/98, 81-451 Gdynia, Poland;1. KU Leuven, Rega Institute for Medical Research, Herestraat 49, Box 1030, 3000 Leuven, Belgium;2. University College London, Department of Structural and Molecular Biology, Gower Street, London, WC1E 6BT, UK |
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| Abstract: | The family B DNA polymerase gene from the euryarchaeon Thermococcus waiotapuensis (Twa) contains an open reading frame of 4404 bases that encodes 1467 amino acid residues. The gene is split by two intein-coding sequences that forms a continuous open reading frame with the three polymerase exteins. Twa DNA polymerase genes with (whole gene) and without (genetically intein-spliced) inteins were expressed in Escherichia coli Rosetta(DE3)pLysS. The inteins of the expressed whole gene were easily spliced during purification. The molecular mass of the purified Twa DNA polymerase was about 90 kDa, as estimated by SDS-PAGE. The optimal pH for Twa DNA polymerase activity was 6.0 and the optimal temperature was 75 °C. The enzyme was activated by magnesium ions. The half-life of the enzyme at 99 °C was about 4 h. The optimal buffer for PCR with Twa DNA polymerase was 50 mM Tris–HCl (pH 8.2), 2.0 mM MgCl2, 30 mM KCl, 2.0 mM (NH4)2SO4, 0.01% Triton X-100, and 0.005% BSA. The PCR fidelity of Twa DNA polymerase was higher than Pfu, KOD and Vent DNA polymerases. A ratio of 15:1 Taq:Twa DNA polymerase efficiently facilitated long-range PCR. |
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