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dc.contributor.authorYamashita, Mami
dc.contributor.authorXu, Jian
dc.contributor.authorMorokuma, Daisuke
dc.contributor.authorHirata, Kazuma
dc.contributor.authorHino, Masato
dc.contributor.authorMon, Hiroaki
dc.contributor.authorTakahashi, Masateru
dc.contributor.authorHamdan, Samir
dc.contributor.authorSakashita, Kosuke
dc.contributor.authorIiyama, Kazuhiro
dc.contributor.authorBanno, Yutaka
dc.contributor.authorKusakabe, Takahiro
dc.contributor.authorLee, Jae Man
dc.date.accessioned2017-05-10T11:18:07Z
dc.date.available2017-05-10T11:18:07Z
dc.date.issued2017-05-08
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.
dc.identifier.issn1073-6085
dc.identifier.issn1559-0305
dc.identifier.pmid28484957
dc.identifier.doi10.1007/s12033-017-0008-9
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.
dc.description.sponsorshipWe thank Dr. Imanishi (National Institute of Agrobiological Sciences, Japan) for providing the NIAS-Bm-oyanagi2 (BmO2) cell line.
dc.publisherSpringer Nature
dc.relation.urlhttp://link.springer.com/article/10.1007/s12033-017-0008-9
dc.rightsThe final publication is available at Springer via http://dx.doi.org/10.1007/s12033-017-0008-9
dc.subjectDNA polymerase
dc.subjectKOD
dc.subjectSilkworm
dc.subjectBaculovirus expression system
dc.titleCharacterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System
dc.typeArticle
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Division
dc.contributor.departmentBioscience Core Lab
dc.contributor.departmentBioscience Program
dc.contributor.departmentLaboratory of DNA Replication and Recombination
dc.contributor.departmentProteomics
dc.identifier.journalMolecular Biotechnology
dc.eprint.versionPost-print
dc.contributor.institutionLaboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Fukuoka, Japan
dc.contributor.institutionLaboratory of Insect Pathology and Microbial Control, Institute of Biological Control, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan
dc.contributor.institutionLaboratory of Silkworm Genetic Resources, Institute of Genetic Resources, Graduate School of Bio Resources and Bioenvironmental Science, Kyushu University, Fukuoka, Japan
kaust.personTakahashi, Masateru
kaust.personHamdan, Samir
kaust.personSakashita, Kosuke
refterms.dateFOA2018-05-08T00:00:00Z
dc.date.published-online2017-05-08
dc.date.published-print2017-06


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