Recombinant thermostable AP exonuclease from Thermoanaerobacter tengcongensis: cloning, expression, purification, properties and PCR application

SÅ‚awomir DÄ…browski, Anna Brillowska-Dabrowska, Birgitte K. Ahring
Pol J Microbiol
2013; 62 (2):
ICID: 1055705
Article type: Original article
IC™ Value: 10.00
Abstract provided by Publisher
 
Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity, especially at high temperatures. The homologue of AP exonuclease from thermophilic anaerobic bacterium Thermoanaerobacter tengcongensis into Escherichia coli was cloned and showed high homology (80%) to human Ape1 nuclease, whereas to E. coli exonuclease III - 78%. This is the fist prokaryotic AP nuclease that exhibited such high homology to human Ape1 nuclease. The very high expression level (57 % of total soluble proteins) of fully active and soluble His6-tagged Tte AP enzyme with His6-domain on C-terminal end was obtained in Escherichia coli Rosetta (DE3) pLysS. The active enzyme was purified up to 98% homogeneity in one chromatographic step using metal-affinity chromatography on Ni2+-IDA-Sepharose resin. A 90 mg (14000 kU) of pure His6-tagged Tte AP (153 kU/mg) from 1 liter of culture was obtained. The optimal conditions (temperature, Mg2+ concentration) of Tte AP endo- , exonuclease and 3’-nuclease activity were investigated using fluorescein labeled dsDNA with inserted AP sites and ssDNA. The optimal Tte AP endonuclease activity was observed at 70-75°C, pH 8.0 and at low Mg2+ concentration (0.5 mM). The higher Mg2+ concentration (>1 mM) enhanced of 3’-5’ exonuclease activity and at Mg2+ concentration >2.0 mM the 3’ nuclease activity was observed. Because of 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 it improved efficiency of the DNA amplification.
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