Abstract
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.
Highlights
With advances in science, there is a growing use of thermostable DNA polymerases
The gene encoding the Stoffel fragment of a Taq DNA polymerase was cloned into the vector pET-30 Ek/LIC to generate a pET30/TaqS plasmid which led to the expression of the enzyme as a protein with a C-terminal polyhistidine tag
The PCR products of the taq Stoffel and NeqSSBlike genes were mixed together with the DNA of the pET-30 Ek/LIC vector in order to obtain the pET30/NeqSSB-TaqS plasmid which encodes the enzyme as a fusion protein containing the complete NeqSSB-like sequence, a 6 amino acid linker (GGVDMI) and a sequence of the Taq Stoffel DNA polymerase with a C-terminal polyhistidine tag
Summary
There is a growing use of thermostable DNA polymerases. Many new enzymes have been identified and described, predominantly those which were isolated from genera such as Thermus, Thermococcus and Pyrococcus. Taq DNA polymerase isolated from the thermophilic eubacterium Thermus aquaticus [1] has revolutionized molecular biology and has become one of the most commonly used polymerases. It is the first thermostable enzyme ever used in PCR [2]. The native Taq DNA polymerase is isolated from Thermus aquaticus and its recombinant form is manufactured commercially using E.coli as a host It has a relatively short half-life as compared to other thermostable polymerases isolated from Archaea. Taq DNA polymerases become completely inhibited when a PCR mixture contains 0.004% of blood [7] It seems that hemoglobin and lactoferrin play an important role in the inhibition of the amplification process. BSA was found to be the most efficient amplification facilitator [8]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
