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

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.

Fig 1

The expression and purification of TaqS (A) and NeqSSB-TaqS (B) DNA polymerases. The proteins were analyzed on a 10% polyacrylamide gel (SDS-PAGE). Lane M: Unstained Protein Weight Marker (Fermentas, Lithuania), molecular masses highlighted. Lane 1: the sonicated extract of induced cells; Lane 2: heat treatment; Lane 3: a by-product after the second washing with the use of the buffer B; Lane 4: purified protein after elution with the buffer C.

Fig 2. Characterization of a fusion NeqSSB-TaqS DNA polymerase in comparison to a TaqS DNA polymerase.

The effect of (A) MgCl₂, (B) KCl, (C) (NH₄)₂SO₄, (D) pH and (E) temperature on the polymerase activity. The results for the NeqSSB-TaqS DNA polymerase are marked with black circles, whilst for the TaqS DNA polymerase with black tringles. Error bars for the TaqS DNA polymerase have the end bar whilst for the Neq-TaqS DNA polymerase does not have the end bar.

Fig 3. Amplification efficiency for DNA polymerases depending on the composition of salt in PCR.

Differences in the amplification efficiency for the fusion NeqSSB-TaqS DNA polymerase (A) and TaqS DNA polymerase (B) depending on the composition of salt in the PCR buffer (10 mM KCl plus 0; 10; 20; 30; or 40 mM of (NH₄)₂SO₄. Lane M: the DNA molecular size marker HyperLadder II (Bioline, UK).

Fig 4. Evaluation of PCR amplification rate.

Comparison of the PCR amplification rates of a fusion NeqSSB-TaqS DNA polymerase for 300 bp (A), 500 bp (B), 1000 bp (C) products and a TaqS DNA polymerase for 300 bp (D), 500 bp (E), 1000 bp (F) products. The elongation times used for the PCR amplification are indicated at the top. Lane M: the DNA molecular size marker (50–2000 bp).

Fig 5. Determination of processivity based on the melting temperatures of DNA products created in the presence of a heparin trap.

(A) Melting curves of the resulting products for the DNA polymerases. (B) Melting temperature of the elongated products.

Fig 6. The primer-template binding for NeqSSB-TaqS and TaqS DNA polymerases.

The binding of fusion NeqSSB-TaqS (A) and native TaqS (B) DNA polymerases to a primer-template measured at various annealing PCR temperatures (indicated in each lane at the top of the gels). The amplified products were analyzed on a 2% agarose gel stained with ethidium bromide.

Fig 7. DNA polymerase tolerance to PCR inhibitors.

The effect of blood (A), lactoferrin (B) and heparin (C) inhibitors on DNA amplification with the use of the genomic DNA of S.aureus as a template and primers for specific nuc gene detection. The effect of whole human blood on the DNA amplification with the use of primers for the amplification of a human CCR5 gene (D). No inhibitors were used in control reactions. Lane M: DNA standards ladder (100–1000 bp). The amplified products were analyzed on a 2% agarose gel stained with ethidium bromide.

Fig 8

A mobility shift assay for TaqS DNA polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT)₇₆ and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT)₇₆ and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT)₇₆ and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT)₇₆ and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT)₇₆ and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol NeqSSB-TaqS DNA polymerase, respectively.