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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
- Śpibida, Marta, Krawczyk, Beata, Zalewska-Piątek, Beata, Piątek, Rafał, Wysocka, Magdalena, Olszewski, Marcin
- Applied microbiology and biotechnology 2018 v.102 no.2 pp. 713-721
- DNA, DNA-binding domains, DNA-directed DNA polymerase, Escherichia coli, Pyrococcus furiosus, half life, heparin, lactoferrin, molecular cloning, polymerase chain reaction, recombinant fusion proteins, thermal stability
- 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.