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

Handle URI:
http://hdl.handle.net/10754/623465
Title:
Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System
Authors:
Yamashita, Mami; Xu, Jian; Morokuma, Daisuke; Hirata, Kazuma; Hino, Masato; Mon, Hiroaki; Takahashi, Masateru; Hamdan, Samir ( 0000-0001-5192-1852 ) ; Sakashita, Kosuke ( 0000-0002-7118-5511 ) ; Iiyama, Kazuhiro; Banno, Yutaka; Kusakabe, Takahiro; Lee, Jae Man
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.
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division; Laboratory of DNA Replication and Recombination; Biosciences Core Lab
Citation:
Yamashita M, Xu J, Morokuma D, Hirata K, Hino M, et al. (2017) Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System. Molecular Biotechnology. Available: http://dx.doi.org/10.1007/s12033-017-0008-9.
Publisher:
Springer Nature
Journal:
Molecular Biotechnology
Issue Date:
8-May-2017
DOI:
10.1007/s12033-017-0008-9
Type:
Article
ISSN:
1073-6085; 1559-0305
Sponsors:
We thank Dr. Imanishi (National Institute of Agrobiological Sciences, Japan) for providing the NIAS-Bm-oyanagi2 (BmO2) cell line.
Additional Links:
http://link.springer.com/article/10.1007/s12033-017-0008-9
Appears in Collections:
Articles; Bioscience Core Lab; Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.authorYamashita, Mamien
dc.contributor.authorXu, Jianen
dc.contributor.authorMorokuma, Daisukeen
dc.contributor.authorHirata, Kazumaen
dc.contributor.authorHino, Masatoen
dc.contributor.authorMon, Hiroakien
dc.contributor.authorTakahashi, Masateruen
dc.contributor.authorHamdan, Samiren
dc.contributor.authorSakashita, Kosukeen
dc.contributor.authorIiyama, Kazuhiroen
dc.contributor.authorBanno, Yutakaen
dc.contributor.authorKusakabe, Takahiroen
dc.contributor.authorLee, Jae Manen
dc.date.accessioned2017-05-10T11:18:07Z-
dc.date.available2017-05-10T11:18:07Z-
dc.date.issued2017-05-08en
dc.identifier.citationYamashita M, Xu J, Morokuma D, Hirata K, Hino M, et al. (2017) Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System. Molecular Biotechnology. Available: http://dx.doi.org/10.1007/s12033-017-0008-9.en
dc.identifier.issn1073-6085en
dc.identifier.issn1559-0305en
dc.identifier.doi10.1007/s12033-017-0008-9en
dc.identifier.urihttp://hdl.handle.net/10754/623465-
dc.description.abstractThe 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.en
dc.description.sponsorshipWe thank Dr. Imanishi (National Institute of Agrobiological Sciences, Japan) for providing the NIAS-Bm-oyanagi2 (BmO2) cell line.en
dc.publisherSpringer Natureen
dc.relation.urlhttp://link.springer.com/article/10.1007/s12033-017-0008-9en
dc.rightsThe final publication is available at Springer via http://dx.doi.org/10.1007/s12033-017-0008-9en
dc.subjectDNA polymeraseen
dc.subjectKODen
dc.subjectSilkwormen
dc.subjectBaculovirus expression systemen
dc.titleCharacterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector Systemen
dc.typeArticleen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
dc.contributor.departmentLaboratory of DNA Replication and Recombinationen
dc.contributor.departmentBiosciences Core Laben
dc.identifier.journalMolecular Biotechnologyen
dc.eprint.versionPost-printen
dc.contributor.institutionLaboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Fukuoka, Japanen
dc.contributor.institutionLaboratory of Insect Pathology and Microbial Control, Institute of Biological Control, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japanen
dc.contributor.institutionLaboratory of Silkworm Genetic Resources, Institute of Genetic Resources, Graduate School of Bio Resources and Bioenvironmental Science, Kyushu University, Fukuoka, Japanen
kaust.authorTakahashi, Masateruen
kaust.authorHamdan, Samiren
kaust.authorSakashita, Kosukeen
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