Fusion of DNA-binding domain of Pyrococcus furiosus ligase with TaqStoffel DNA polymerase as a useful tool in PCR with difficult targets

Marta Śpibida,Beata Zalewska-Piątek,Beata Krawczyk,Marcin Olszewski,Rafał Piątek,Magdalena Wysocka

doi:10.1007/s00253-017-8560-6

Marta Śpibida, Beata Zalewska-Piątek + Show 4 more

Open Access

https://doi.org/10.1007/s00253-017-8560-6

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Abstract

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.

Highlights

At the core of PCR-based techniques is the use of a DNA polymerase which plays the major role in molecular biological applications including DNA amplification or sequencing (Terpe 2013)
PCR requires a high temperature, and it is necessary to use a thermostable enzyme for DNA amplification; the most commonly used enzyme is a DNA polymerase derived from the thermophilic Thermus aquaticus
The PCR experiment was performed with the use of 1 U of purified PfuDBDlig-TaqStoffel DNA polymerase (TaqS) DNA polymerase and with the use of TaqS DNA polymerase which were placed in a 20 μl reaction mixture containing 50 ng of the E. coli genomic DNA as a template

Summary

Introduction

At the core of PCR-based techniques is the use of a DNA polymerase which plays the major role in molecular biological applications including DNA amplification or sequencing (Terpe 2013). PCR requires a high temperature, and it is necessary to use a thermostable enzyme for DNA amplification; the most commonly used enzyme is a DNA polymerase derived from the thermophilic Thermus aquaticus. The Taq DNA polymerase is not able to synthesize products which are longer than 4 kb (Hamilton et al 2001). A novel strategy used to enhance the processivity and performance of the Taq is to fuse it with a thermostable DNA-binding protein such as a Sso7d DNAbinding protein derived from Sulfolobus solfataricus (Wang et al 2004)

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