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

被引：15

作者：

Yamashita, Mami ^{[1
]}

Xu, Jian ^{[1
]}

Morokuma, Daisuke ^{[1
]}

Hirata, Kazuma ^{[1
]}

Hino, Masato ^{[1
]}

Mon, Hiroaki ^{[1
]}

Takahashi, Masateru ^{[2
]}

Hamdan, Samir M. ^{[2
]}

Sakashita, Kosuke ^{[3
]}

Iiyama, Kazuhiro ^{[4
]}

Banno, Yutaka ^{[5
]}

Kusakabe, Takahiro ^{[1
]}

Lee, Jae Man ^{[1
]}

机构：

[1] Kyushu Univ, Grad Sch Bioresource & Bioenvironm Sci, Lab Insect Genome Sci, Higashi Ku, 6-10-1 Hakozaki, Fukuoka 8128581, Japan

[2] King Abdullah Univ Sci & Technol, Lab DNA Replicat & Recombinat, Div Biol & Environm Sci & Engn, 4700 KAUST Thuwal, Jeddah 23955, Saudi Arabia

[3] King Abdullah Univ Sci & Technol, Biosci Core Lab, Prote, 4700 KAUST Thuwal, Jeddah 23955, Saudi Arabia

[4] Kyushu Univ, Lab Insect Pathol & Microbial Control, Inst Biol Control, Fac Agr,Grad Sch,Higashi Ku, Hakozaki 6-10-1, Fukuoka 8128581, Japan

[5] Kyushu Univ, Inst Genet Resources, Grad Sch Bio Resources & Bioenvironm Sci, Lab Silkworm Genet Resources,Higashi Ku, Hakozaki 6-10-1, Fukuoka 8128581, Japan

来源：

MOLECULAR BIOTECHNOLOGY | 2017年 / 59卷 / 06期

关键词：

DNA polymerase; KOD; Silkworm; Baculovirus expression system; PYROCOCCUS-FURIOSUS; ESCHERICHIA-COLI; CHAIN-REACTION; PCR; EFFICIENT; FIDELITY; PURIFICATION; PERFORMANCE; STRATEGY; PROTEIN;

D O I：

10.1007/s12033-017-0008-9

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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 degrees 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.

引用

页码：221 / 233

页数：13

共 42 条

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