Cloning, expression, and characterization of thermostable DNA polymerase from Thermoanaerobacter yonseiensis.
J Biochem Mol Biol
; 35(3): 320-9, 2002 May 31.
Article
em En
| MEDLINE
| ID: mdl-12297016
ABSTRACT
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
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Base de dados:
MEDLINE
Assunto principal:
Pyrococcus
/
DNA Polimerase Dirigida por DNA
/
Genes Bacterianos
Idioma:
En
Ano de publicação:
2002
Tipo de documento:
Article