Cloning, Purification, and Characterization of a New DNA Polymerase from a Hyperthermophilic Archaeon, Thermococcus sp. NA1

Genomic analysis of Thermococcus sp. NA1 revealed the presence of a 3,927-base-pair (bp) family B-type DNA polymerase gene, TNA1_pol. TNA1_pol, without its intein, was overexpressed in Escherichia coli, purified using metal affinity chromatography, and characterized. TNA1_pol activity was optimal at...

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Veröffentlicht in:	Journal of microbiology and biotechnology 2007-07, Vol.17 (7), p.1090-1097
Hauptverfasser:	Kim, Y.J. (Korea Ocean Research and Development Institute, Ansan, Republic of Korea), Lee, H.S. (Korea Ocean Research and Development Institute, Ansan, Republic of Korea), Bae, S.S. (Korea Ocean Research and Development Institute, Ansan, Republic of Korea), Jeon, J.H. (Korea Ocean Research and Development Institute, Ansan, Republic of Korea), Lim, J.K. (Korea Ocean Research and Development Institute, Ansan, Republic of Korea), Cho, Y.A. (Korea Ocean Research and Development Institute, Ansan, Republic of Korea), Nam, K.H. (Sungkyunkwan University, Suwon, Republic of Korea), Kang, S.G. (Korea Ocean Research and Development Institute, Ansan, Republic of Korea), Kim, S.J. (Korea Ocean Research and Development Institute, Ansan, Republic of Korea), Kwon, S.T. (Sungkyunkwan University, Suwon, Republic of Korea), Lee, J.H. (Korea Ocean Research and Development Institute, Ansan, Republic of Korea), E-mail: jlee@kordi.re.kr
Format:	Artikel
Sprache:	eng
Schlagworte:	ADN Amino Acid Motifs Amino Acid Sequence Archaea - enzymology Archaeal Proteins - chemistry Archaeal Proteins - genetics Archaeal Proteins - metabolism Base Sequence Biological and medical sciences Biotechnology characterization Cloning, Molecular DNA DNA Primers - chemistry DNA Primers - metabolism DNA, Bacterial - genetics DNA, Bacterial - isolation & purification DNA-Directed DNA Polymerase - analysis DNA-Directed DNA Polymerase - chemistry DNA-Directed DNA Polymerase - genetics DNA-Directed DNA Polymerase - isolation & purification Enzyme Stability Escherichia coli - genetics family B-type DNA polymerase fidelity Fundamental and applied biological sciences. Psychology Genes, Bacterial Half-Life Hydrogen-Ion Concentration hyperthermophile Molecular Sequence Data Molecular Weight Open Reading Frames Polymerase Chain Reaction processivity Protein Structure, Tertiary Recombinant Proteins - chemistry Recombinant Proteins - metabolism Sequence Analysis, DNA Temperature Thermococcus - enzymology Thermococcus - genetics
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Zusammenfassung:	Genomic analysis of Thermococcus sp. NA1 revealed the presence of a 3,927-base-pair (bp) family B-type DNA polymerase gene, TNA1_pol. TNA1_pol, without its intein, was overexpressed in Escherichia coli, purified using metal affinity chromatography, and characterized. TNA1_pol activity was optimal at pH 7.5 and 75℃. TNA1_pol was highly thermostable, with a half-life of 3.5 h at 100℃ and 12.5 h at 95℃. Polymerase chain reaction parameters of TNA1_pol such as error-rate, processivity, and extension rate were measured in comparison with rTaq, Pfu, and KOD DNA polymerases.
ISSN:	1017-7825