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

Mami Yamashita, Jian Xu, Daisuke Morokuma, Kazuma Hirata, Masato Hino, Hiroaki Mon, Masateru Takahashi, Samir M. Hamdan, Kosuke Sakashita, Kazuhiro Iiyama, Yutaka Banno, Takahiro Kusakabe, Man Lee

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)221-233
Number of pages13
JournalMolecular Biotechnology
Volume59
Issue number6
DOIs
Publication statusPublished - Jun 1 2017

Fingerprint

Thermococcus
Bombyx
Baculoviridae
DNA-Directed DNA Polymerase
Larva
DNA
Polymerase Chain Reaction
Escherichia coli
Elongation
Shock
Fusion reactions
Hot Temperature

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology

Cite this

Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System. / Yamashita, Mami; Xu, Jian; Morokuma, Daisuke; Hirata, Kazuma; Hino, Masato; Mon, Hiroaki; Takahashi, Masateru; Hamdan, Samir M.; Sakashita, Kosuke; Iiyama, Kazuhiro; Banno, Yutaka; Kusakabe, Takahiro; Lee, Man.

In: Molecular Biotechnology, Vol. 59, No. 6, 01.06.2017, p. 221-233.

Research output: Contribution to journalArticle

Yamashita, Mami ; Xu, Jian ; Morokuma, Daisuke ; Hirata, Kazuma ; Hino, Masato ; Mon, Hiroaki ; Takahashi, Masateru ; Hamdan, Samir M. ; Sakashita, Kosuke ; Iiyama, Kazuhiro ; Banno, Yutaka ; Kusakabe, Takahiro ; Lee, Man. / Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System. In: Molecular Biotechnology. 2017 ; Vol. 59, No. 6. pp. 221-233.
@article{d3646937b6554eeab3599afe0d5040e2,
title = "Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System",
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.",
author = "Mami Yamashita and Jian Xu and Daisuke Morokuma and Kazuma Hirata and Masato Hino and Hiroaki Mon and Masateru Takahashi and Hamdan, {Samir M.} and Kosuke Sakashita and Kazuhiro Iiyama and Yutaka Banno and Takahiro Kusakabe and Man Lee",
year = "2017",
month = "6",
day = "1",
doi = "10.1007/s12033-017-0008-9",
language = "English",
volume = "59",
pages = "221--233",
journal = "Molecular Biotechnology",
issn = "1073-6085",
publisher = "Humana Press",
number = "6",

}

TY - JOUR

T1 - Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System

AU - Yamashita, Mami

AU - Xu, Jian

AU - Morokuma, Daisuke

AU - Hirata, Kazuma

AU - Hino, Masato

AU - Mon, Hiroaki

AU - Takahashi, Masateru

AU - Hamdan, Samir M.

AU - Sakashita, Kosuke

AU - Iiyama, Kazuhiro

AU - Banno, Yutaka

AU - Kusakabe, Takahiro

AU - Lee, Man

PY - 2017/6/1

Y1 - 2017/6/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=85019038034&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85019038034&partnerID=8YFLogxK

U2 - 10.1007/s12033-017-0008-9

DO - 10.1007/s12033-017-0008-9

M3 - Article

VL - 59

SP - 221

EP - 233

JO - Molecular Biotechnology

JF - Molecular Biotechnology

SN - 1073-6085

IS - 6

ER -