RRC ID 50402
著者 Yamashita M, Xu J, Morokuma D, Hirata K, Hino M, Mon H, Takahashi M, Hamdan SM, Sakashita K, Iiyama K, Banno Y, Kusakabe T, Lee JM.
タイトル Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System.
ジャーナル Mol Biotechnol
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.
巻・号 59(6)
ページ 221-233
公開日 2017-6-1
DOI 10.1007/s12033-017-0008-9
PII 10.1007/s12033-017-0008-9
PMID 28484957
MeSH Animals Bombyx / genetics* Genetic Vectors / genetics* Recombinant Proteins / genetics* Thermococcus / genetics*
IF 2.022
引用数 8
リソース情報
カイコ n70