Cloning, Expression, and Characterization of Family A DNA Polymerase from <i>Massilia aurea</i>
<i>Mau</i> DNA polymerase is a family A DNA polymerase isolated from <i>Massilia aurea</i>. In this study, a recombinant plasmid, His<sub>6</sub>-tagged Mau-pET28c, was constructed. His-tagged <i>Mau</i> was expressed in <i>Escherichia coli</i...
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MDPI AG
2023-07-01
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| Series: | Fermentation |
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| Online Access: | https://www.mdpi.com/2311-5637/9/7/650 |
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| author | Aleksandra A. Kuznetsova Ksenia S. Bedritskikh Anatoly A. Bulygin Nikita A. Kuznetsov |
| author_facet | Aleksandra A. Kuznetsova Ksenia S. Bedritskikh Anatoly A. Bulygin Nikita A. Kuznetsov |
| author_sort | Aleksandra A. Kuznetsova |
| collection | DOAJ |
| description | <i>Mau</i> DNA polymerase is a family A DNA polymerase isolated from <i>Massilia aurea</i>. In this study, a recombinant plasmid, His<sub>6</sub>-tagged Mau-pET28c, was constructed. His-tagged <i>Mau</i> was expressed in <i>Escherichia coli</i> Rosseta 2 (DE3) competent cells and, after optimization of purification conditions, was successfully isolated via a two-step purification system by Ni<sup>2+</sup>-chelating affinity chromatography followed by heparin affinity chromatography. The biochemical properties of <i>Mau</i> DNA polymerase were investigated next. This polymerase showed maximal polymerase activity at 30 °C, pH 8.4–8.8, 2–10 mM MgCl<sub>2</sub>, and 10–40 mM KCl. Kinetic parameters of correct and incorrect dNTP incorporation as well as DNA-binding affinity were determined too. <i>K</i><sup>dNTP</sup><sub>d,app</sub> values were found to be 16 µM for correct dNTP and 200–500 µM for incorrect dNTP. The kinetic parameter <i>k</i><sub>cat</sub> turned out to be 0.2 s<sup>−1</sup> for correct dNTP incorporation and an order of magnitude less for incorrect dNTP incorporation. It was demonstrated that <i>Mau</i> DNA polymerase has 5′→3′ and 3′→5′ exonuclease activities associated with the main activity. |
| first_indexed | 2024-03-11T01:05:53Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 2311-5637 |
| language | English |
| last_indexed | 2024-03-11T01:05:53Z |
| publishDate | 2023-07-01 |
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| record_format | Article |
| series | Fermentation |
| spelling | doaj.art-a0f0d18d22da46dfa825f9765ff872762023-11-18T19:16:27ZengMDPI AGFermentation2311-56372023-07-019765010.3390/fermentation9070650Cloning, Expression, and Characterization of Family A DNA Polymerase from <i>Massilia aurea</i>Aleksandra A. Kuznetsova0Ksenia S. Bedritskikh1Anatoly A. Bulygin2Nikita A. Kuznetsov3Institute of Chemical Biology and Fundamental Medicine (ICBFM), Siberian Branch of Russian Academy of Sciences (SB RAS), 8 Prospekt Akad. Lavrentyeva, Novosibirsk 630090, RussiaDepartment of Natural Sciences, Novosibirsk State University, 2 Pirogova Str., Novosibirsk 630090, RussiaInstitute of Chemical Biology and Fundamental Medicine (ICBFM), Siberian Branch of Russian Academy of Sciences (SB RAS), 8 Prospekt Akad. Lavrentyeva, Novosibirsk 630090, RussiaInstitute of Chemical Biology and Fundamental Medicine (ICBFM), Siberian Branch of Russian Academy of Sciences (SB RAS), 8 Prospekt Akad. Lavrentyeva, Novosibirsk 630090, Russia<i>Mau</i> DNA polymerase is a family A DNA polymerase isolated from <i>Massilia aurea</i>. In this study, a recombinant plasmid, His<sub>6</sub>-tagged Mau-pET28c, was constructed. His-tagged <i>Mau</i> was expressed in <i>Escherichia coli</i> Rosseta 2 (DE3) competent cells and, after optimization of purification conditions, was successfully isolated via a two-step purification system by Ni<sup>2+</sup>-chelating affinity chromatography followed by heparin affinity chromatography. The biochemical properties of <i>Mau</i> DNA polymerase were investigated next. This polymerase showed maximal polymerase activity at 30 °C, pH 8.4–8.8, 2–10 mM MgCl<sub>2</sub>, and 10–40 mM KCl. Kinetic parameters of correct and incorrect dNTP incorporation as well as DNA-binding affinity were determined too. <i>K</i><sup>dNTP</sup><sub>d,app</sub> values were found to be 16 µM for correct dNTP and 200–500 µM for incorrect dNTP. The kinetic parameter <i>k</i><sub>cat</sub> turned out to be 0.2 s<sup>−1</sup> for correct dNTP incorporation and an order of magnitude less for incorrect dNTP incorporation. It was demonstrated that <i>Mau</i> DNA polymerase has 5′→3′ and 3′→5′ exonuclease activities associated with the main activity.https://www.mdpi.com/2311-5637/9/7/650DNA polymerase<i>Massilia aurea</i>enzyme activityfamily Anative enzyme |
| spellingShingle | Aleksandra A. Kuznetsova Ksenia S. Bedritskikh Anatoly A. Bulygin Nikita A. Kuznetsov Cloning, Expression, and Characterization of Family A DNA Polymerase from <i>Massilia aurea</i> Fermentation DNA polymerase <i>Massilia aurea</i> enzyme activity family A native enzyme |
| title | Cloning, Expression, and Characterization of Family A DNA Polymerase from <i>Massilia aurea</i> |
| title_full | Cloning, Expression, and Characterization of Family A DNA Polymerase from <i>Massilia aurea</i> |
| title_fullStr | Cloning, Expression, and Characterization of Family A DNA Polymerase from <i>Massilia aurea</i> |
| title_full_unstemmed | Cloning, Expression, and Characterization of Family A DNA Polymerase from <i>Massilia aurea</i> |
| title_short | Cloning, Expression, and Characterization of Family A DNA Polymerase from <i>Massilia aurea</i> |
| title_sort | cloning expression and characterization of family a dna polymerase from i massilia aurea i |
| topic | DNA polymerase <i>Massilia aurea</i> enzyme activity family A native enzyme |
| url | https://www.mdpi.com/2311-5637/9/7/650 |
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