Dietrich Jacques, Schmitt Philippe, Zieger Montserrat, Preve Brigitte, Rolland Jean-Luc, Chaabihi Hassan, Gueguen Yannick
Ifremer, Direction des Ressources Vivantes, France.
FEMS Microbiol Lett. 2002 Nov 19;217(1):89-94. doi: 10.1111/j.1574-6968.2002.tb11460.x.
DNA polymerase from the archaeon Pyrococcus abyssi strain Orsay was expressed in Escherichia coli. The recombinant DNA polymerase (Pab) was purified to homogeneity by heat treatment followed by 5 steps of chromatography and characterized for PCR applications. Buffer optimization experiments indicated that Pab PCR performance and fidelity parameters were highest in the presence of 20 mM Tris-HCl, pH 9.0, 1.5 mM MgSO4, 25 mM KCl, 10 mM (NH4)2SO4 and 40 microM of each dNTP. Under these conditions, the error rate was 0.66.10(-6) mutations/nucleotide/duplication. Pab DNA polymerase, having a half life of 5 h at 100 degrees C, was demonstrated to be highly thermostable in PCR conditions compared to commercial Taq and Pfu DNA polymerases. These characteristics enable Pab to be one of the most efficient thermostable DNA polymerases described, exhibiting very high accuracy compared to other available commercial DNA polymerases and robust thermostable activity. This new DNA polymerase is currently on the market under the name Isis DNA Polymerase (Qbiogene Molecular Biology).
来自深海热球菌奥尔赛菌株（Pyrococcus abyssi strain Orsay）的DNA聚合酶在大肠杆菌中表达。重组DNA聚合酶（Pab）通过热处理，随后经过5步色谱法纯化至同质，并针对PCR应用进行了表征。缓冲液优化实验表明，在含有20 mM Tris-HCl（pH 9.0）、1.5 mM MgSO4、25 mM KCl、10 mM (NH4)2SO4和40 μM各dNTP的情况下，Pab的PCR性能和保真度参数最高。在这些条件下，错误率为0.66×10⁻⁶突变/核苷酸/复制。与市售的Taq和Pfu DNA聚合酶相比，Pab DNA聚合酶在100℃下半衰期为5小时，在PCR条件下表现出高度的热稳定性。这些特性使Pab成为所描述的最有效的热稳定DNA聚合酶之一，与其他市售DNA聚合酶相比具有非常高的准确性和强大的热稳定活性。这种新的DNA聚合酶目前以Isis DNA聚合酶（Qbiogene分子生物学公司）的名称投放市场。
