Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Rapid and Simple Method to Prepare Functional Pfu DNA Polymerase Expressed in Escherichia coli Periplasm

  • Chae, Young-Kee (Department of Applied Chemistry and Recombinant Protein Expression Center (RPEC), Sejong University) ;
  • Jeon, Woo-Chun (Department of Applied Chemistry and Recombinant Protein Expression Center (RPEC), Sejong University) ;
  • Cho, Kyoung-Suk (Department of Applied Chemistry and Recombinant Protein Expression Center (RPEC), Sejong University)
  • Published : 2002.10.01
Download PDF
⟨ Previous Next ⟩

Abstract

Pfu DNA polymerase from Pyrococcus furiosus was expressed in the E. coli periplasm, and the fully active polymerase was partially purified by applying osmotic shock, ammonium sulfate precipitation, and heat treatment. This method represents a new way of expressing and purifying functional Pfu DNA polymerase without the use of chromatography.

Keywords

References

  1. Proc. Natl. Acad. Sci. USA v.91 PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Barnes, W. M. https://doi.org/10.1073/pnas.91.6.2216
  2. Nucleic Acids Res. v.24 PCR fidelity of Pfu DNA polymerase and other other thermostable DNA polymerases. Cline, J.;J. C. Braman;H. H. Hogrefe. https://doi.org/10.1093/nar/24.18.3546
  3. Protein Expr. Purif. v.14 Cloning and expression in Escherichia coli of the recombinant His-Tagges DNA polymerases from Pyrococcus furiosus and Pyrcoccus woesei. Dabrowski, S.;J. Kur. https://doi.org/10.1006/prep.1998.0945
  4. PCR Methods Appl. v.1 DNA polymerase fidelity and the polymerase chain reaction. Eckert, K. A.;T. A. Kunkel. https://doi.org/10.1101/gr.1.1.17
  5. Nucleic Acids Res. v.22 A rapid PCR fidelity assay. Flaman, J. M.;T. Frebourg;V. Moreau;F. Charbonnier;C. Martin;C. Ishioka;S. H. Friend;R. Iggo. https://doi.org/10.1093/nar/22.15.3259
  6. J. Microbiol. Biotechnol. v.11 Cloning ad sequencing of the β-amylase gene from Paenibacillus sp. and its expression in Saccharomyces cerevisiae. Jeong, T.-H.;H.-O. Kim;J.-N. Park;H. Lee;D.-J. Shin;H. Blaise Lee;S. B. Chun;S. Bai.
  7. J. Biol. Chem. v.268 Characterizatioin of a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis. Vent DNA polymerase, steady state kinetics, thermal stability, processivity, strand displacement, and exonuclease activities. Kong, H.;R. B. Kucera;W. E. Jack.
  8. Biochemistry v.35 Exonucelopytic proofreading during replication of repetitive DNA. Kroutil, L. C.;K. Register;K. Bebenek;T. A. Kunkel. https://doi.org/10.1021/bi952178h
  9. Protein Expr. Purif. v.11 Expression in Escherichia coli of the thermostable DNA polymerase from Pyrococcus furiosus. Lu, C.;H. P. Erickson. https://doi.org/10.1006/prep.1997.0775
  10. Gene v.108 High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Lundberg, K. S.;D. D. Shoemaker;W. M. Adams;J. M. Short;J. A. Sorge;E. J. Mathur. https://doi.org/10.1016/0378-1119(91)90480-Y
  11. J. Microbiol. Biotechnol. v.11 Prediction of the secondary structure of the AfgA subunit of Salmonella enteritidis overexpressed as an MBP-fused protein. Won, M.;S. Kim;S. Lee;C. Kim;H. Kim;M. Jun;K. B. Song.