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Fusion of DNA-binding domain of Pyrococcus furiosus ligase with TaqStoffel DNA polymerase as a useful tool in PCR with difficult targets
Abstract
The DNA coding sequence of TaqStoffel polymer- ase 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 Taq DNA 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.
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- Category:
- Articles
- Type:
- artykuł w czasopiśmie wyróżnionym w JCR
- Published in:
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APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
no. 102,
pages 713 - 721,
ISSN: 0175-7598 - Language:
- English
- Publication year:
- 2018
- Bibliographic description:
- Śpibida M., Krawczyk B., Zalewska-Piątek B., Piątek R., Wysocka M., Olszewski M.: Fusion of DNA-binding domain of Pyrococcus furiosus ligase with TaqStoffel DNA polymerase as a useful tool in PCR with difficult targets// APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. -Vol. 102, nr. 2 (2018), s.713-721
- DOI:
- Digital Object Identifier (open in new tab) 10.1007/s00253-017-8560-6
- Verified by:
- Gdańsk University of Technology
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