Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR
Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR
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DOI：
10.1128/aem.63.11.4504-4510.1997
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发表时间：
1997-11-01
影响因子：
4.4
通讯作者：
Imanaka, T
中科院分区：
文献类型：
--
作者：
Takagi, M;Nishioka, M;Imanaka, T
古细菌Pyrococcus sp.菌株KOD 1的DNA聚合酶基因（KOD DNA聚合酶）含有5，013个碱基的长开放阅读框，编码1，671个氨基酸残基（GenBank登录号D29671），相似性分析揭示DNA聚合酶含有推定的3 '-5'外切核酸酶活性和两个框内插入序列1，080 bp（360个氨基酸; ROD pol内含肽-1）和1，611 bp（537个氨基酸; KOD pol内含肽-2），它们位于真核生物和古细菌α-样DNA聚合酶之间保守区域的中间。成熟形式的DNA聚合酶基因在大肠杆菌中表达，并对重组酶进行纯化和表征。证实了3 '-5'核酸外切酶活性，虽然KOD DNA聚合酶的最适温度（75 ℃）和突变频率（3.5 × 10 ~（-3））与来自激烈火球菌的DNA聚合酶相似（Pfu DNA聚合酶），KOD DNA聚合酶表现出延伸速率（100至130个核苷酸/秒）高5倍和持续合成能力（连续核苷酸聚合的持续性）高10至15倍。这些特性使得KOD DNA聚合酶能够在更短的反应时间内进行更准确的PCR。
The DNA polymerase gene from the archaeon Pyrococcus sp, strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671), Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; ROD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conserved among eukaryotic and archaeal alpha-like DNA polymerases. The mature form of the DNA polymerase gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. 3'-5' exonuclease activity was confirmed, and although KOD DNA polymerase's optimum temperature (75 degrees C) and mutation frequency (3.5 x 10(-3)) were similar to those of a DNA polymerase from Pyrococcus furiosus (Pfu DNA polymerase), the KOD DNA polymerase exhibited an extension rate (100 to 130 nucleotides/s) 5 times higher and a processivity (persistence of sequential nucleotide polymerization) 10 to 15 times higher than those of Pfu DNA polymerase . These characteristics enabled the KOD DNA polymerase to perform a more accurate PCR in a shorter reaction time.