Cloning, expression, and characterization of thermostable DNA polymerase from Thermoanaerobacter yonseiensis.
A gene, coined tay, for a thermostable DNA polymerase from the novel, extremely thermophilic bacterium Thermoanaerobacter yonseiensis was cloned and expressed in E. coli. Using a DNA polymerase homologous PCR product as a hybridization probe, tay was isolated and sequenced to consist of 2,616 nucleotides that encode 872 amino acids. A database analysis showed that DNA polymerase, coined Tay, from T. yonseiensis shared a 39 percent to 47 percent identity in the amino acid sequence with those from other DNA polymerases. Tay was overexpressed in E. coli as a fusion protein with a poly-histidine tag at the Cterminus. It was purified by heat treatment, followed by a Ni(2+)-chelate column. The molecular weight of purified Tay was approximately 97 kDa, as shown by SDS PAGE, and it showed high DNA polymerase activity and thermostability. However, it had no 3'-->5' exonuclease activity
Kim
Dae Jin
DJ
Programs in Biomaterials Science and Engineering, College of Science, Yonsei University, Seoul 120-749, Korea.
Jang
Hyeung Jin
HJ
Pyun
Yu Ryang
YR
Kim
Yu Sam
YS
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004261:DNA Replication; D004269:DNA, Bacterial; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D019711:Pyrococcus / Q000201:enzymology / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004269:DNA, Bacterial; D008670:Metals; D004259:DNA-Directed DNA Polymerase