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Construction, Expression, and Characterization of Recombinant Pfu DNA Polymerase in Escherichia coli

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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 Ni2+ 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.

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Abbreviations

AP:

Ammonium persulfate

bp:

Base pair

E. coli :

Escherichia coli

EDTA:

Ethylene diamine tetraacetic acid

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

PAGE:

Polyacrylamide gel electrophoresis

pEGFP:

Plasmid enhanced green fluorescent protein

pI:

Isoelectric point

PMSF:

Phenylmethylsulfonyl fluoride

SDS:

Sodium dodecyl sulfate

TEMED:

N,N,N′,N′-Tetramethylethylenediamine

U:

Unit

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Acknowledgments

We would like to thank the Natural Basic Sciences Personnel Training Foundation of China (No. J1310033) for funding support. We acknowledged Professor Jijie Chai (National Institute of Biological Sciences of Beijing) for the provision of Pfu gene.

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Authors and Affiliations

  1. College of Life Sciences, Peking University, Beijing, 100871, People’s Republic of China

    Wenjun Zheng, Qingsong Wang & Qun Bi

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  1. Wenjun Zheng
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  2. Qingsong Wang
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  3. Qun Bi
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Correspondence to Qun Bi.

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Zheng, W., Wang, Q. & Bi, Q. Construction, Expression, and Characterization of Recombinant Pfu DNA Polymerase in Escherichia coli . Protein J 35, 145–153 (2016). https://doi.org/10.1007/s10930-016-9651-4

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