Abstract
Genomic analysis of Thermococcus sp. NA revealed the presence of a 3,927-base-pair (bp) family B-type DNA polymerase gene, TNA1_pol. TNA1_pol, without its intein, was overexpressed in Escherichia coli, purified using metal affinity chromatography, and characterized. TNA1_pol activity was optimal at pH 7.5 and 75 degrees C. TNA1_pol was highly thermostable, with a half-life of 3.5 h at 100 degrees C and 12.5 h at 95 degrees C. Polymerase chain reaction parameters of TNA1_pol such as error-rate, processivity, and extension rate were measured in comparison with rTaq, Pfu, and KOD DNA polymerases. TNA1_pol averaged one incorrect bp every 4.45 kilobases (kb), and had a processivity of 150 nucleotides (nt) and an extension rate of 60 bases/s. Thus, TNA1_pol has a much faster elongation rate than Pfu DNA polymerase with 7-fold higher fidelity than that of rTaq.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Motifs
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Amino Acid Sequence
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Archaea / enzymology*
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Archaeal Proteins / chemistry
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Archaeal Proteins / genetics
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Archaeal Proteins / metabolism
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Base Sequence
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Cloning, Molecular
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DNA Primers / chemistry
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DNA Primers / metabolism
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DNA, Bacterial / genetics
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DNA, Bacterial / isolation & purification
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DNA-Directed DNA Polymerase / analysis
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DNA-Directed DNA Polymerase / chemistry
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DNA-Directed DNA Polymerase / genetics*
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DNA-Directed DNA Polymerase / isolation & purification*
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Enzyme Stability
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Escherichia coli / genetics
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Genes, Bacterial
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Half-Life
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Hydrogen-Ion Concentration
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Molecular Sequence Data
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Molecular Weight
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Open Reading Frames
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Polymerase Chain Reaction
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Protein Structure, Tertiary
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Sequence Analysis, DNA
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Temperature
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Thermococcus / enzymology*
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Thermococcus / genetics
Substances
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Archaeal Proteins
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DNA Primers
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DNA, Bacterial
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Recombinant Proteins
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DNA-Directed DNA Polymerase