Expression and Purification of Pfu DNA Polymerase Belonging to the B Family Polymerase

Document Type : Original Article (s)

Authors

1 PharmD Student, School of Pharmacy and Pharmaceutical Sciences AND Student Research Committee, Isfahan University of Medical Sciences, Isfahan, Iran

2 Associate Professor, Department of Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

3 Department of Agricultural Biotechnology, School of Agriculture, Isfahan University of Technology, Isfahan, Iran

4 Assistant Professor, Department of Biochemistry AND Bioinformatics Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract

Background: DNA polymerases are enzymes directing the synthesis of DNA molecules from deoxyribonucleotides. They are essential tools for molecular biology. Pfu DNA polymerase was initially isolated from Pyrococcus furiosus, anaerobic hyperthermophilic archaeon lived in geothermally heated marine sediments with temperatures between 90°C and 100°C. This enzyme possesses 3´ to 5´ exonucleotic activity; so that makes correcting the errors made in DNA replication.Methods: In this research, the DNA fragment encoding Pfu DNA polymerase was cloned in to pET-15b expression vector. Then, by changing expression conditions such as isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, and time and temperature of the induction, the expression of this enzyme was optimized. Finally, Pfu DNA polymerase was produced in large scale in optimized conditions and purified with simple method. Then, purified enzyme was used in polymerase chain reaction (PCR) for evaluating the activity.Findings: Transformation of recombinant vector produced some colonies that most of them have the plasmid. The expression of Pfu DNA polymerase resulted in a bond approximately 90 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Maximum amount of protein production was observed in IPTG concentration of 0.75 mM, at 37°C, 3 hours incubation.Conclusion: Protein purification with using the method based on Desai protocol caused a product that had the activity like commercial one in PCR reaction.

Keywords

References

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Journal of Isfahan Medical School
Volume 31, Issue 251 - Serial Number 251
July and August 2013
Pages 1392-1404

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History

  • Receive Date: 26 June 2013
  • Revise Date: 06 May 2024
  • Accept Date: 06 April 2022

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