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
AU - Sakashita, Kosuke
AU - Iiyama, Kazuhiro
AU - Banno, Yutaka
AU - Kusakabe, Takahiro
AU - Lee, Jae Man
N1 - KAUST Repository Item: Exported on 2020-10-01
Acknowledgements: We thank Dr. Imanishi (National Institute of Agrobiological Sciences, Japan) for providing the NIAS-Bm-oyanagi2 (BmO2) cell line.
PY - 2017/5/8
Y1 - 2017/5/8
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://hdl.handle.net/10754/623465
UR - http://link.springer.com/article/10.1007/s12033-017-0008-9
UR - http://www.scopus.com/inward/record.url?scp=85019038034&partnerID=8YFLogxK
U2 - 10.1007/s12033-017-0008-9
DO - 10.1007/s12033-017-0008-9
M3 - Article
C2 - 28484957
SN - 1073-6085
VL - 59
SP - 221
EP - 233
JO - Molecular Biotechnology
JF - Molecular Biotechnology
IS - 6
ER -