Cloning, purification, and characterization of a new DNA polymerase from a hyperthermophilic archaeon, Thermococcus sp. NA1.
Cloning, purification, and characterization of a new DNA polymerase from a hyperthermophilic archaeon, Thermococcus sp. NA1.
复制标题
DOI：
--
复制
发表时间：
2007-07
影响因子：
2.8
通讯作者：
Y. Kim;H. Lee;S. S. Bae-S.;J. Jeon;J. Lim;Yona Cho;K. Nam;S. Kang;Sang-Jin Kim;S. Kwon;Jung-Hyun Lee
中科院分区：
文献类型：
--
作者：
Y. Kim;H. Lee;S. S. Bae-S.;J. Jeon;J. Lim;Yona Cho;K. Nam;S. Kang;Sang-Jin Kim;S. Kwon;Jung-Hyun Lee
NA的基因组分析显示存在3，927个碱基对（bp）的家族B型DNA聚合酶基因TNA1_pol。TNA1_pol不含内含肽，在大肠杆菌中过表达，使用金属亲和层析纯化，并表征。TNA1_pol的最适pH值为7.5，最适温度为75 ℃。TNA1_pol具有高度的热稳定性，在100 ℃下的半衰期为3.5小时，在95 ℃下为12.5小时。与rTaq，Pfu和KOD DNA聚合酶相比，TNA1_pol的聚合酶链反应参数，如错误率，持续合成能力和延伸率进行了测量。TNA1_pol平均每4.45个碱基（kb）就有一个错误的bp，并且具有150个核苷酸（nt）的持续合成能力和60个碱基/秒的延伸速率。因此，TNA1_pol具有比Pfu DNA聚合酶快得多的延伸速率，具有比rTaq高7倍的保真度。
Genomic analysis of Thermococcus sp. NA 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 degrees C. TNA1_pol was highly thermostable, with a half-life of 3.5 h at 100 degrees C and 12.5 h at 95 degrees C. 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. TNA1_pol averaged one incorrect bp every 4.45 kilobases (kb), and had a processivity of 150 nucleotides (nt) and an extension rate of 60 bases/s. Thus, TNA1_pol has a much faster elongation rate than Pfu DNA polymerase with 7-fold higher fidelity than that of rTaq.