Cloning, expression, and characterization of a family B-type DNA polymerase from the hyperthermophilic crenarchaeon Pyrobaculum arsenaticum and its application to PCR SCIE SCOPUS KCI

Cited 14 time in WEB OF SCIENCE Cited 15 time in Scopus
Title
Cloning, expression, and characterization of a family B-type DNA polymerase from the hyperthermophilic crenarchaeon Pyrobaculum arsenaticum and its application to PCR
Author(s)
Shin, HJ; Lee, SK; Choi, JJ; Koh, S; Lee, JH; Kim, SJ; Kwon, ST
KIOST Author(s)
Lee, Jung Hyun(이정현)
Publication Year
2005-12
Abstract
The gene encoding Pyrobaculum arsenaticum DNA polymerase (Par DNA polymerase) was cloned and sequenced. The gene consists of 2,361 bp coding for a protein with 786 amino acid residues. The deduced amino acid sequence of Par DNA polymerase showed a high similarity to archaeal family 13-type DNA polymerases (Group I), and contained all of the motifs conserved in the family 13-type DNA polymerases for 3'-> 5' exonuclease and polymerase activities. The Par DNA polymerase gene was expressed under the control of the T7lac promoter on the expression vector pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RP. The expressed enzyme was purified by heat treatment, and Cibacron blue 3GA and HiTrap (TM) Heparin HP column chromatographies. The optimum pH of the purified enzyme was 7.5. The enzyme activity was activated by divalent cations, and was inhibited by EDTA and monovalent cations. The half-life of the enzyme at 95 degrees C was 6 h. Par DNA polymerase possessed associated 3'-> 5' proofreading exonuclease activity, which is consistent with its deduced amino acid sequence. PCR experiment with Par DNA polymerase showed an amplified product, indicating that this enzyme might be useful in DNA amplification and PCR-based applications.
ISSN
1017-7825
URI
https://sciwatch.kiost.ac.kr/handle/2020.kiost/5003
Bibliographic Citation
JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, v.15, no.6, pp.1359 - 1367, 2005
Publisher
KOREAN SOC MICROBIOLOGY & BIOTECHNOLOGY
Subject
THERMUS-CALDOPHILUS GK24; HIGH-LEVEL EXPRESSION; DEOXYRIBONUCLEIC-ACID; PYROCOCCUS-FURIOSUS; NUCLEOTIDE-SEQUENCE; ARCHAEA; PURIFICATION; ENZYME; GENE; AMPLIFICATION
Keywords
Archaea; DNA polymerase; exonuclease activity; polymerase chain reaction; Pyrobaculum arsenaticum; thermostable enzyme
Type
Article
Language
English
Document Type
Article
Publisher
KOREAN SOC MICROBIOLOGY & BIOTECHNOLOGY
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