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

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

• DNA-dependent DNA polymerase
• Polymerase chain reaction (PCR)
• Cloning
• pET-15b