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

Igor P. Oscorbin, Ekaterina A. Belousova, Ulyana A. Boyarskikh, Aleksandr I. Zakabunin, Evgeny A. Khrapov, Maksim L. Filipenko

    Research output: Contribution to journalArticlepeer-review

    7 Citations (Scopus)

    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 DNAbinding 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.

    Original languageEnglish
    Pages (from-to)9595-9610
    Number of pages16
    JournalNucleic Acids Research
    Volume45
    Issue number16
    DOIs
    Publication statusPublished - 19 Sep 2017

    Keywords

    • Catalytic Domain
    • Cloning, Molecular
    • DNA/metabolism
    • DNA Polymerase I/antagonists & inhibitors
    • Enzyme Inhibitors/pharmacology
    • Genome, Human
    • Geobacillus/enzymology
    • Geobacillus stearothermophilus/enzymology
    • Humans
    • Nucleic Acid Amplification Techniques/methods
    • Protein Engineering/methods
    • Protein Stability
    • Pyrococcus abyssi/genetics
    • Recombinant Fusion Proteins/genetics
    • Sulfolobus/genetics

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