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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
Abstrakt
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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Informacje szczegółowe
- Kategoria:
- Publikacja w czasopiśmie
- Typ:
- artykuł w czasopiśmie wyróżnionym w JCR
- Opublikowano w:
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APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
nr 102,
strony 713 - 721,
ISSN: 0175-7598 - Język:
- angielski
- Rok wydania:
- 2018
- Opis bibliograficzny:
- 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:
- Cyfrowy identyfikator dokumentu elektronicznego (otwiera się w nowej karcie) 10.1007/s00253-017-8560-6
- Weryfikacja:
- Politechnika Gdańska
wyświetlono 158 razy