Abstract
The gene encoding Aquifex aeolicus (Aae) DNA polymerase was expressed under the control of the trp promoter on a high-copy plasmid, pTRPNS, in Escherichia coli. The expressed enzyme was purified 11-fold with a 13.8% yield and a specific activity of 2268.3 U mg(-1). The optimum pH of the enzyme was 6.8-7.2. The optimal concentrations of KCl and Mg(2+) were 20-30 mM and 4-5 mM, respectively. Aae DNA polymerase contained a double-strand-dependent 3'-->5' proofreading exonuclease activity but lacked any detectable 5'-->3' exonuclease activity.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Cations, Divalent / pharmacology
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Cloning, Molecular
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DNA / metabolism
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DNA-Directed DNA Polymerase / genetics
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DNA-Directed DNA Polymerase / isolation & purification*
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DNA-Directed DNA Polymerase / metabolism*
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Edetic Acid / pharmacology
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Escherichia coli / genetics*
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Exonucleases / metabolism
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Gram-Negative Aerobic Rods and Cocci / enzymology*
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Gram-Negative Aerobic Rods and Cocci / genetics
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Hydrogen-Ion Concentration
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Magnesium / pharmacology
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Potassium Chloride / pharmacology
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Temperature
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Transformation, Bacterial
Substances
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Cations, Divalent
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Recombinant Proteins
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Potassium Chloride
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DNA
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Edetic Acid
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DNA-Directed DNA Polymerase
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Exonucleases
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Magnesium