Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization - Publikacja - MOST Wiedzy

Twoje konto

zaloguj

Wyszukiwarka

Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization

Abstrakt

DNA polymerases are present in all organisms and are important enzymes that synthesise DNA molecules. They are used in various fields of science, predominantly as essential components for in vitro DNA syntheses, known as PCR. Modern diagnostics, molecular biology and genetic engineering need DNA polymerases which demonstrate improved performance. This study was aimed at obtaining a new NeqSSB-TaqS fusion DNA polymerase from the Taq DNA Stoffel domain and a single-stranded DNA binding-like protein of Nanoarchaeum equitans in order to significantly improve the properties of DNA polymerase. The DNA coding sequence of Taq Stoffel DNA polymerase and the nonspecific DNA-binding protein of Nanoarchaeum equitans (NeqSSB-like protein) were fused. A novel recombinant gene was obtained which was cloned into the pET-30 Ek/LIC vector and introduced into E. coli for expression. The recombinant enzyme was purified and its enzymatic properties including DNA polymerase activity, PCR amplification rate, thermostability, processivity and resistance to inhibitors, were tested. The yield of the target protein reached approximately 18 mg/l after 24 h of the IPTG induction. The specific activity of the polymerase was 2200 U/mg. The recombinant NeqSSB-TaqS exhibited a much higher extension rate (1000 bp template in 20 s), processivity (19 nt), thermostability (half-life 35 min at 95°C) and higher tolerance to PCR inhibitors (0.3-1.25% of whole blood, 0.84-13.5 μg of lactoferrin and 4.7-150 ng of heparin) than Taq Stoffel DNA polymerase. Furthermore, our studies show that NeqSSB-TaqS DNA polymerase has a high level of flexibility in relation to Mg2+ ions (from 1 to 5 mM) and KCl or (NH4)2SO4 salts (more than 60 mM and 40 mM, respectively). Using NeqSSB-TaqS DNA polymerase instead of the Taq DNA polymerase could be a better choice in many PCR applications.

Cytowania

  • 1 1

    CrossRef

  • 0

    Web of Science

  • 1 0

    Scopus

Autorzy (4)

Cytuj jako

Pełna treść

pobierz publikację
pobrano 64 razy
Wersja publikacji
Accepted albo Published Version
DOI:
Cyfrowy identyfikator dokumentu elektronicznego (otwiera się w nowej karcie) 10.1371/journal.pone.0184162
Licencja
Creative Commons: CC-BY otwiera się w nowej karcie

Słowa kluczowe

Informacje szczegółowe

Kategoria:
Publikacja w czasopiśmie
Typ:
artykuł w czasopiśmie wyróżnionym w JCR
Opublikowano w:
PLOS ONE nr 12, wydanie 9, strony 1 - 17,
ISSN: 1932-6203
Język:
angielski
Rok wydania:
2017
Opis bibliograficzny:
Olszewski M., Śpibida M., Bilek M., Krawczyk B.: Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization// PLOS ONE. -Vol. 12, iss. 9 (2017), s.1-17
DOI:
Cyfrowy identyfikator dokumentu elektronicznego (otwiera się w nowej karcie) 10.1371/journal.pone.0184162
Weryfikacja:
Politechnika Gdańska

wyświetlono 171 razy

Publikacje, które mogą cię zainteresować

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

2018

Taq DNA polymerase fused with DNA binding protein with increased resistance to inhibitors from clinical samples

2015

Modified DNA polymerases for PCR troubleshooting

2017

Increased concentration of Taq DNA polymerase as a solution for GC-rich templates from clinical and environmental samples

2015
Meta Tagi