title: Construction, Expression, and Characterization of Recombinant Pfu DNA Polymerase in Escherichia coli.
abstract: 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.
authors.affiliation: College of Life Sciences, Peking University, Beijing, 100871, People's Republic of China.
authors.forename: Wenjun
authors.initials: W
authors.lastname: Zheng
authors.affiliation: College of Life Sciences, Peking University, Beijing, 100871, People's Republic of China.
authors.forename: Qingsong
authors.initials: Q
authors.lastname: Wang
authors.affiliation: College of Life Sciences, Peking University, Beijing, 100871, People's Republic of China. biqunb@pku.edu.cn.
authors.forename: Qun
authors.initials: Q
authors.lastname: Bi
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D007526:Isoelectric Point; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase
keywords: Isoelectric point; Peptide mass fingerprint; Pfu DNA polymerase; Polymerase chain reaction