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Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System

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Abstract

The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.

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Acknowledgements

We thank Dr. Imanishi (National Institute of Agrobiological Sciences, Japan) for providing the NIAS-Bm-oyanagi2 (BmO2) cell line.

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Authors and Affiliations

  1. Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki Higashi-ku, Fukuoka, 812-8581, Japan

    Mami Yamashita, Jian Xu, Daisuke Morokuma, Kazuma Hirata, Masato Hino, Hiroaki Mon, Takahiro Kusakabe & Jae Man Lee

  2. Laboratory of DNA Replication and Recombination, Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, 4700 KAUST Thuwal, Jeddah, 23955, Saudi Arabia

    Masateru Takahashi & Samir M. Hamdan

  3. Bioscience Core Lab, Proteomics, King Abdullah University of Science and Technology, 4700 KAUST Thuwal, Jeddah, 23955, Saudi Arabia

    Kosuke Sakashita

  4. Laboratory of Insect Pathology and Microbial Control, Institute of Biological Control, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812-8581, Japan

    Kazuhiro Iiyama

  5. Laboratory of Silkworm Genetic Resources, Institute of Genetic Resources, Graduate School of Bio Resources and Bioenvironmental Science, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812-8581, Japan

    Yutaka Banno

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  1. Mami Yamashita
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Yamashita, M., Xu, J., Morokuma, D. et al. Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System. Mol Biotechnol 59, 221–233 (2017). https://doi.org/10.1007/s12033-017-0008-9

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