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 |