New DNA polymerase from the hyperthermophilic marine archaeon Thermococcus thioreducens.
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.
Marsic
Damien
D
HudsonAlpha Institute for Biotechnology, 601 Genome Way, Huntsville, AL 35806, USA.
Flaman
Jean-Michel
JM
Ng
Joseph D
JD
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D011994:Recombinant Proteins; D004259:DNA-Directed DNA Polymerase