Zheng Wenjun, Wang Qingsong, Bi Qun
College of Life Sciences, Peking University, Beijing, 100871, People's Republic of China.
Protein J. 2016 Apr;35(2):145-53. doi: 10.1007/s10930-016-9651-4.
Pfu DNA polymerase (Pfu) is a DNA polymerase isolated from the hyperthermophilic archaeon Pyrococcus furiosus. With its excellent thermostability and high fidelity, Pfu is well known as one of the enzymes widely used in the polymerase chain reaction. In this study, the recombinant plasmid pLysS His6-tagged Pfu-pET28a was constructed. His-tagged Pfu was expressed in Escherichia coli BL21 (DE3) competent cells and then successfully purified with the ÄKTAprime plus compact one-step purification system by Ni(2+) chelating affinity chromatography after optimization of the purification conditions. The authenticity of the purified Pfu was further confirmed by peptide mass fingerprinting. A bio-assay indicated that its activity in the polymerase chain reaction was equivalent to that of commercial Pfu and its isoelectric point was found to be between 6.85 and 7.35. These results will be useful for further studies on Pfu and its wide application in the future.
Pfu DNA聚合酶（Pfu）是一种从嗜热古菌激烈火球菌中分离出来的DNA聚合酶。凭借其出色的热稳定性和高保真度，Pfu是聚合酶链反应中广泛使用的酶之一。在本研究中，构建了重组质粒pLysS His6标记的Pfu-pET28a。His标记的Pfu在大肠杆菌BL21（DE3）感受态细胞中表达，然后在优化纯化条件后，通过Ni(2+)螯合亲和色谱法，使用ÄKTAprime plus紧凑型一步纯化系统成功纯化。通过肽质量指纹图谱进一步确认了纯化后的Pfu的真实性。生物测定表明，其在聚合酶链反应中的活性与市售Pfu相当，并且发现其等电点在6.85至7.35之间。这些结果将有助于未来对Pfu的进一步研究及其广泛应用。
