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Recombinant !ermostable AP Exonuclease from Thermoanaerobacter tengcongensis: Cloning, Expression, Purification, Properties and PCR Application
Abstract
Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity, especially at high temperatures. !e gene encoding a homologue of AP exonuclease was cloned from the thermophilic anaerobic bacterium Thermoanaerobacter tengcongensis and transformed into Escherichia coli. The protein product showed high identity (80%) to human Ape1 nuclease, whereas to E. coli exonuclease III – 78%. This is the first prokaryotic AP nuclease that exhibits such high identity to human Ape1 nuclease. !e very high expression level (57% of total soluble proteins) of fully active and soluble His6-tagged Tte AP enzyme with His6-tag on C-terminal end was obtained in Escherichia coli Rosetta (DE3) pLysS. !e active enzyme was purified up to 98% homogeneity in one chromatographic step using metal-affinity chromatography on Ni2+ -IDA-Sepharose resin. !e yield was 90 mg (14 000 kU) of pure His6-tagged Tte AP (153kU/mg) from 1 liter of culture. The optimal conditions of Tte AP endo-, exonuclease and 3’-nuclease activity were investigated using fluorescein labeled dsDNA with inserted AP sites and ssDNA. Optimal Tte AP endonuclease activity was observed at 70–75°C, pH 8.0 and at low Mg2+ concentration (0.5 mM). Higher Mg2+ concentration (> 1 mM) enhanced 3’-5’ exonuclease activity and at Mg2+ concentration > 2.0 mM 3’ nuclease activity was observed. Because of the endonuclease activity of Tte AP exonuclease, the enzyme was applied in PCR amplification of long DNA templates. Tte AP exonuclease eliminated AP-sites in DNA template and improved the efficiency of DNA amplification.
Authors (3)
-
Sławomir Dąbrowski dr inż.
- DTU e Environmental Microbiology & Biotechnology Research Group, BioCentrum,
-
Birgitte Ahring
- DTU e Environmental Microbiology & Biotechnology Research Group, BioCentrum,
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- Category:
- Articles
- Type:
- artykuł w czasopiśmie wyróżnionym w JCR
- Published in:
-
Polish Journal of Microbiology
no. 62,
pages 121 - 129,
ISSN: 1733-1331 - Language:
- English
- Publication year:
- 2013
- Bibliographic description:
- Dąbrowski S., Brillowska-Dąbrowska A., Ahring B.: Recombinant !ermostable AP Exonuclease from Thermoanaerobacter tengcongensis: Cloning, Expression, Purification, Properties and PCR Application // Polish Journal of Microbiology. -Vol. 62, nr. 2 (2013), s.121-129
- Verified by:
- Gdańsk University of Technology
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