Standard

Cloning, Expression, and Characterization of Family A DNA Polymerase from Massilia aurea. / Kuznetsova, Aleksandra A.; Bedritskikh, Ksenia S.; Bulygin, Anatoly A. et al.

In: Fermentation, Vol. 9, No. 7, 650, 07.2023.

Research output: Contribution to journal › Article › peer-review

Harvard

Kuznetsova, AA, Bedritskikh, KS, Bulygin, AA & Kuznetsov, NA 2023, 'Cloning, Expression, and Characterization of Family A DNA Polymerase from Massilia aurea', Fermentation, vol. 9, no. 7, 650. https://doi.org/10.3390/fermentation9070650

APA

Kuznetsova, A. A., Bedritskikh, K. S., Bulygin, A. A., & Kuznetsov, N. A. (2023). Cloning, Expression, and Characterization of Family A DNA Polymerase from Massilia aurea. Fermentation, 9(7), [650]. https://doi.org/10.3390/fermentation9070650

Vancouver

Kuznetsova AA, Bedritskikh KS, Bulygin AA, Kuznetsov NA. Cloning, Expression, and Characterization of Family A DNA Polymerase from Massilia aurea. Fermentation. 2023 Jul;9(7):650. doi: 10.3390/fermentation9070650

Author

Kuznetsova, Aleksandra A. ; Bedritskikh, Ksenia S. ; Bulygin, Anatoly A. et al. / Cloning, Expression, and Characterization of Family A DNA Polymerase from Massilia aurea. In: Fermentation. 2023 ; Vol. 9, No. 7.

BibTeX

@article{3f58f5c79e60489c9433cadbe8320bfb,
title = "Cloning, Expression, and Characterization of Family A DNA Polymerase from Massilia aurea",
abstract = "Mau DNA polymerase is a family A DNA polymerase isolated from Massilia aurea. In this study, a recombinant plasmid, His6-tagged Mau-pET28c, was constructed. His-tagged Mau was expressed in Escherichia coli Rosseta 2 (DE3) competent cells and, after optimization of purification conditions, was successfully isolated via a two-step purification system by Ni2+-chelating affinity chromatography followed by heparin affinity chromatography. The biochemical properties of Mau DNA polymerase were investigated next. This polymerase showed maximal polymerase activity at 30 °C, pH 8.4–8.8, 2–10 mM MgCl2, and 10–40 mM KCl. Kinetic parameters of correct and incorrect dNTP incorporation as well as DNA-binding affinity were determined too. KdNTPd,app values were found to be 16 µM for correct dNTP and 200–500 µM for incorrect dNTP. The kinetic parameter kcat turned out to be 0.2 s−1 for correct dNTP incorporation and an order of magnitude less for incorrect dNTP incorporation. It was demonstrated that Mau DNA polymerase has 5′→3′ and 3′→5′ exonuclease activities associated with the main activity.",
keywords = "DNA polymerase, Massilia aurea, enzyme activity, family A, native enzyme",
author = "Kuznetsova, {Aleksandra A.} and Bedritskikh, {Ksenia S.} and Bulygin, {Anatoly A.} and Kuznetsov, {Nikita A.}",
note = "This research was funded by the Ministry of Science and Higher Education under agreement No. 075-15-2021-1085.",
year = "2023",
month = jul,
doi = "10.3390/fermentation9070650",
language = "English",
volume = "9",
journal = "Fermentation",
issn = "2311-5637",
publisher = "Multidisciplinary Digital Publishing Institute (MDPI)",
number = "7",

}

RIS

TY - JOUR

T1 - Cloning, Expression, and Characterization of Family A DNA Polymerase from Massilia aurea

AU - Kuznetsova, Aleksandra A.

AU - Bedritskikh, Ksenia S.

AU - Bulygin, Anatoly A.

AU - Kuznetsov, Nikita A.

N1 - This research was funded by the Ministry of Science and Higher Education under agreement No. 075-15-2021-1085.

PY - 2023/7

Y1 - 2023/7

N2 - Mau DNA polymerase is a family A DNA polymerase isolated from Massilia aurea. In this study, a recombinant plasmid, His6-tagged Mau-pET28c, was constructed. His-tagged Mau was expressed in Escherichia coli Rosseta 2 (DE3) competent cells and, after optimization of purification conditions, was successfully isolated via a two-step purification system by Ni2+-chelating affinity chromatography followed by heparin affinity chromatography. The biochemical properties of Mau DNA polymerase were investigated next. This polymerase showed maximal polymerase activity at 30 °C, pH 8.4–8.8, 2–10 mM MgCl2, and 10–40 mM KCl. Kinetic parameters of correct and incorrect dNTP incorporation as well as DNA-binding affinity were determined too. KdNTPd,app values were found to be 16 µM for correct dNTP and 200–500 µM for incorrect dNTP. The kinetic parameter kcat turned out to be 0.2 s−1 for correct dNTP incorporation and an order of magnitude less for incorrect dNTP incorporation. It was demonstrated that Mau DNA polymerase has 5′→3′ and 3′→5′ exonuclease activities associated with the main activity.

AB - Mau DNA polymerase is a family A DNA polymerase isolated from Massilia aurea. In this study, a recombinant plasmid, His6-tagged Mau-pET28c, was constructed. His-tagged Mau was expressed in Escherichia coli Rosseta 2 (DE3) competent cells and, after optimization of purification conditions, was successfully isolated via a two-step purification system by Ni2+-chelating affinity chromatography followed by heparin affinity chromatography. The biochemical properties of Mau DNA polymerase were investigated next. This polymerase showed maximal polymerase activity at 30 °C, pH 8.4–8.8, 2–10 mM MgCl2, and 10–40 mM KCl. Kinetic parameters of correct and incorrect dNTP incorporation as well as DNA-binding affinity were determined too. KdNTPd,app values were found to be 16 µM for correct dNTP and 200–500 µM for incorrect dNTP. The kinetic parameter kcat turned out to be 0.2 s−1 for correct dNTP incorporation and an order of magnitude less for incorrect dNTP incorporation. It was demonstrated that Mau DNA polymerase has 5′→3′ and 3′→5′ exonuclease activities associated with the main activity.

KW - DNA polymerase

KW - Massilia aurea

KW - enzyme activity

KW - family A

KW - native enzyme

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85166441116&origin=inward&txGid=3e73a0bd05f509c91c49d92d889c2830

UR - https://www.mendeley.com/catalogue/05090ac0-0080-34e9-a307-c08c145a3bf5/

U2 - 10.3390/fermentation9070650

DO - 10.3390/fermentation9070650

M3 - Article

VL - 9

JO - Fermentation

JF - Fermentation

SN - 2311-5637

IS - 7

M1 - 650

ER -

ID: 59262618