Результаты исследований: Научные публикации в периодических изданиях › статья › Рецензирование
Derivatives of Bst-like Gss-polymerase with improved processivity and inhibitor tolerance. / Oscorbin, Igor P.; Belousova, Ekaterina A.; Boyarskikh, Ulyana A. и др.
в: Nucleic Acids Research, Том 45, № 16, 19.09.2017, стр. 9595-9610.Результаты исследований: Научные публикации в периодических изданиях › статья › Рецензирование
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TY - JOUR
T1 - Derivatives of Bst-like Gss-polymerase with improved processivity and inhibitor tolerance
AU - Oscorbin, Igor P.
AU - Belousova, Ekaterina A.
AU - Boyarskikh, Ulyana A.
AU - Zakabunin, Aleksandr I.
AU - Khrapov, Evgeny A.
AU - Filipenko, Maksim L.
N1 - © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
PY - 2017/9/19
Y1 - 2017/9/19
N2 - 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.
AB - 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.
KW - Catalytic Domain
KW - Cloning, Molecular
KW - DNA/metabolism
KW - DNA Polymerase I/antagonists & inhibitors
KW - Enzyme Inhibitors/pharmacology
KW - Genome, Human
KW - Geobacillus/enzymology
KW - Geobacillus stearothermophilus/enzymology
KW - Humans
KW - Nucleic Acid Amplification Techniques/methods
KW - Protein Engineering/methods
KW - Protein Stability
KW - Pyrococcus abyssi/genetics
KW - Recombinant Fusion Proteins/genetics
KW - Sulfolobus/genetics
UR - http://www.scopus.com/inward/record.url?scp=85031898426&partnerID=8YFLogxK
U2 - 10.1093/nar/gkx645
DO - 10.1093/nar/gkx645
M3 - Article
C2 - 28934494
AN - SCOPUS:85031898426
VL - 45
SP - 9595
EP - 9610
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 16
ER -
ID: 9875571