New DNA polymerase from the hyperthermophilic marine archaeon <Emphasis Type="Italic">Thermococcus thioreducens</Emphasis> |
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Authors: | Damien Marsic Jean-Michel Flaman Joseph D Ng |
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Institution: | (1) HudsonAlpha Institute for Biotechnology, 601 Genome Way, Huntsville, AL 35806, USA;(2) Laboratory for Structural Biology, Department of Biological Sciences, University of Alabama in Huntsville, Huntsville, AL 35899, USA;(3) INSERM U614, 76183 Rouen, France |
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Abstract: | The family B DNA polymerase gene of Thermococcus thioreducens, an archaeon recently isolated from the Rainbow hydrothermal vent field, was cloned and its protein product expressed, purified
and characterized. The gene was found to encode a 1,311 amino acid chain including an intein sequence of 537 residues. Phylogenetic
analysis revealed a predominantly vertical type of inheritance of the intein in the Thermococcales order. Primary sequence
analysis of the mature protein (TthiPolB) showed significant sequence conservation among DNA polymerases in this family. The
structural fold of TthiPolB was predicted against the known crystallographic structure of a family B DNA polymerase from Thermococcus gorgonarius, allowing regional domain assignments within the TthiPolB sequence. The recombinant TthiPolB was overexpressed in Escherichia coli and purified for biochemical characterization. Compared with other DNA polymerases from the Thermococcales order, TthiPolB
was found to have moderate thermal stability and fidelity, and a high extension rate, consistent with an extremely low K
m
corresponding to the dNTP substrate. TthiPolB performed remarkably well in a wide range of PCR conditions, being faster,
more stable and more accurate than many commonly used enzymes. |
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Keywords: | DNA polymerase Archaea Hyperthermophilic PCR Thermococcus |
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