将 Pyrococcus furiosus 连接酶的 DNA 结合域与 TaqStoffel DNA 聚合酶融合，作为在困难靶标 PCR 中的有用工具。
Fusion of DNA-binding domain of Pyrococcus furiosus ligase with TaqStoffel DNA polymerase as a useful tool in PCR with difficult targets.
机构信息
Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, ul. G. Narutowicza 11/12, 80-233, Gdańsk, Poland.
出版信息
Appl Microbiol Biotechnol. 2018 Jan;102(2):713-721. doi: 10.1007/s00253-017-8560-6. Epub 2017 Nov 4.
The DNA coding sequence of TaqStoffel polymerase was fused with the DNA-binding domain of Pyrococcus furiosus ligase. The resulting novel recombinant gene was cloned and expressed in E. coli. The recombinant enzyme was purified and its enzymatic features were studied. The fusion protein (PfuDBDlig-TaqS) was found to have enhanced processivity as a result of the conversion of the TaqDNA polymerase from a relatively low processive to a highly processive enzyme. The abovementioned processivity enhancement was about threefold as compared to the recombinant TaqStoffel DNA polymerase (TaqS), and the recombinant fusion protein was more thermostable. It had a half-life of 23 min at 99 °C as compared to 10 min for TaqS. The fusion protein also showed a significantly higher resistance to PCR inhibitors such as heparin or lactoferrin and the fusion polymerase-amplified GC-rich templates much more efficiently and was efficient even with 78% GC pairs.
TaqStoffel 聚合酶的 DNA 编码序列与 Pyrococcus furiosus 连接酶的 DNA 结合结构域融合。所得的新型重组基因在大肠杆菌中进行了克隆和表达。对重组酶进行了纯化，并研究了其酶学性质。融合蛋白（PfuDBDlig-TaqS）由于 TaqDNA 聚合酶从相对低的延伸性转变为高延伸性酶，从而表现出增强的延伸性。与重组 TaqStoffel DNA 聚合酶（TaqS）相比，这种延伸性增强约为三倍，并且重组融合蛋白具有更高的热稳定性。它在 99°C 下的半衰期为 23 分钟，而 TaqS 为 10 分钟。融合蛋白还对肝素或乳铁蛋白等 PCR 抑制剂具有更高的抗性，并且融合聚合酶扩增富含 GC 的模板的效率更高，即使 GC 对为 78%也有效。
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