Olszewski Marcin, Śpibida Marta, Bilek Maciej, Krawczyk Beata
Gdańsk University of Technology, Department of Molecular Biotechnology and Microbiology, ul. G. Narutowicza 11/12, Gdańsk, Poland.
Department of Food and Agriculture Production Engineering, University of Rzeszów, ul. Zelwerowicza 4, Rzeszów, Poland.
PLoS One. 2017 Sep 1;12(9):e0184162. doi: 10.1371/journal.pone.0184162. eCollection 2017.
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.
DNA聚合酶存在于所有生物体中，是合成DNA分子的重要酶类。它们在各个科学领域都有应用，主要作为体外DNA合成（即PCR）的必需成分。现代诊断、分子生物学和基因工程需要性能更优的DNA聚合酶。本研究旨在从Taq DNA Stoffel结构域和嗜热栖热菌的一种单链DNA结合样蛋白中获得一种新的NeqSSB - TaqS融合DNA聚合酶，以显著改善DNA聚合酶的性能。将Taq Stoffel DNA聚合酶的DNA编码序列与嗜热栖热菌的非特异性DNA结合蛋白（NeqSSB样蛋白）进行融合。获得了一个新的重组基因，将其克隆到pET - 30 Ek/LIC载体中，并导入大肠杆菌进行表达。对重组酶进行了纯化，并测试了其酶学性质，包括DNA聚合酶活性、PCR扩增速率、热稳定性、持续合成能力和对抑制剂的抗性。IPTG诱导24小时后，目标蛋白产量达到约18 mg/L。聚合酶的比活性为2200 U/mg。重组NeqSSB - TaqS表现出比Taq Stoffel DNA聚合酶更高的延伸速率（20秒内扩增1000 bp模板）、持续合成能力（19个核苷酸）、热稳定性（95°C下半衰期35分钟）以及对PCR抑制剂更高的耐受性（全血的0.3 - 1.25%、乳铁蛋白的0.84 - 13.5 μg和肝素的4.7 - 150 ng）。此外，我们的研究表明，NeqSSB - TaqS DNA聚合酶在Mg2 +离子（1至5 mM）以及KCl或（NH4）2SO4盐（分别超过60 mM和40 mM）方面具有高度的灵活性。在许多PCR应用中，使用NeqSSB - TaqS DNA聚合酶代替Taq DNA聚合酶可能是更好的选择。
