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

Slawomir Dabrowski, Anna Brillowska-Dabrowska, Birgitte Kiær Ahring

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    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. The 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. The 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. The active enzyme was purified up to 98% homogeneity in one chromatographic step using metal-affinity chromatography on Ni(2+)-IDA-Sepharose resin. The yield was 90 mg (14000 kU) of pure His6-tagged Tte AP (153 kU/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 degrees 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.
    Original languageEnglish
    JournalPolish Journal of Microbiology
    Volume62
    Issue number2
    Pages (from-to)121-129
    ISSN1733-1331
    Publication statusPublished - 2013

    Keywords

    • Amino Acid Sequence
    • Bacterial Proteins
    • Cloning, Molecular
    • Exonucleases
    • Gene Expression Regulation, Bacterial
    • Gene Expression Regulation, Enzymologic
    • Molecular Sequence Data
    • Phylogeny
    • Polymerase Chain Reaction
    • Recombinant Proteins
    • Thermoanaerobacter
    • EC 3.1.- Exonucleases

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