Derivatives of Bst-like Gss-polymerase with improved processivity and inhibitor tolerance

Abstract

At the moment, one of the actual trends in medical diagnostics is a development of methods for practical applications such as point-of-care testing, POCT or research tools, for example, whole genome amplification, WGA. All the techniques are based on using of specific DNA polymerases having strand displacement activity, high synthetic processivity, fidelity and, most significantly, tolerance to contaminants, appearing from analysed biological samples or collected under purification procedures. Here, we have designed a set of fusion enzymes based on catalytic domain of DNA polymerase I from Geobacillus sp. 777 with DNA-binding domain of DNA ligase Pyrococcus abyssi and Sto7d protein from Sulfolobus tokodaii, analogue of Sso7d. Designed chimeric DNA polymerases DBD-Gss, Sto-Gss and Gss-Sto exhibited the same level of thermal stability, thermal transferase activity and fidelity as native Gss; however, the processivity was increased up to 3-fold, leading to about 4-fold of DNA product in WGA which is much more exiting. The attachment of DNA-binding proteins enhanced the inhibitor tolerance of chimeric polymerases in loop-mediated isothermal amplification to several of the most common DNA sample contaminants-urea and whole blood, heparin, ethylenediaminetetraacetic acid, NaCl, ethanol. Therefore, chimeric Bst-like Gss-polymerase will be promising tool for both WGA and POCT due to increased processivity and inhibitor tolerance.

Figure 1.

Schematic representation of Gss fusions (A), expression (lanes 1–7) and purification (lanes 8–14) of chimeric proteins (B). Enzymes were expressed in either Escherichia coli strains BL-21 (DE3) pLysS (Gss, Gss-His, DBD-Gss, Gss-DBD, Sto-Gss, Gss-Sto) or XL10-Gold (His-Gss), and purified using affinity and ion-exchange chromatography. M—Precision Plus Protein standards (Bio-Rad, USA), 1, 8—Gss, 2, 9—His-Gss, 3, 10—Gss-His, 4, 11—DBD-Gss, 5, 12—Gss-DBD, 6, 13—Sto-Gss, 7, 14—Gss-Sto. Chimeric proteins are marked by black arrows.

Figure 2.

Thermal shift assay of chimeric polymerases. Thermal denaturation profiles (A) and first derivatives of fluorescent curves (B) are presented; curve color indicates the particular polymerase. Each experiment was triplicated, typical curves are presented.

Figure 3.

DNA-binding capacity of chimeric enzymes. Indicated amounts of enzymes were incubated with either 5′-overhang dsDNA (A) or ssDNA (B) at 55°C. Graphs below present the quantitative analysis of electropherograms.

Figure 4.

Processivity of the chimeric enzymes. Each trace represents one lane from a sequencing gel and each peak represents a single primer extension product. Reaction time was 10 min for each enzyme. The x-axis indicates the primer extension product length, which is determined based on size markers run on the same gel (trace not shown).

Figure 5.

Ion concentrations (A–D), temperature (E) and thermostability (F and G) assays of the chimeric polymerases. Enzyme characteristics were determined via incorporation of α-[³²P] dAMP in activated calf thymus DNA. Each experiment was triplicated. Optimal temperature and thermal stability was determined in standard polymerase activity assays.

Figure 6.

Terminal transferase activity was defined under the incorporation of dNMP on the blunt-ended dsDNA fragment. (A) Kinetics of terminal transferase reaction. Enzymes were incubated for indicated times with DNA substrate and dNTPs at 55°C. The graph below reflects the percent of elongated DNA. (B) Nucleotide preferences under the incorporation of dNMP into blunt-ended DNA.

Figure 7.

Strand displacement activity of the chimeric enzymes. Enzymes were incubated with either primed template or primed template with terminator for indicated times at 55°C. Controls: 1—primed template, 2—primed template with terminator.

Figure 8.

Fidelity of DNA synthesis catalysed by the chimeric enzymes. Template oligonucleotides contained different nucleobases at the +1 position with respect to the 3′-end of the primer. Reactions were performed in the presence of either mixture or one of the dNTPs.

Figure 9.

Fold of WGA with chimeric enzymes. Each WGA sample was analysed in triplicate. A standard curve was generated to determine the locus copy number in amplified DNA relative to genomic DNA at each locus. Fold amplification values were calculated as a ratio of DNA quantity after WGA to initial DNA quantity for each locus.

Figure 10.

Efficacy of ddPCR performed using chimeric DNA polymerases. Each WGA sample was analysed in triplicate. Amplification fold for each locus was calculated as a ratio of DNA quantity after WGA to initial DNA quantity for each locus.

Figure 11.

Robustness of qLAMP with Gss derivatives. qLAMP reactions were conducted using a model system with a Lambda DNA as template and SYTO-82. Resulting time-to-threshold (Tt) values were plotted against concentration of different inhibitor (A-F).