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Cloning, expression and purification of Pwo polymerase from Pyrococcus woesei
Amir Ghasemi1, Ali Hatef Salmanian, Nourkhoda Sadeghifard
1Department of Pathobiology, Institute of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
Background And Objectives:
Pyrococcus woesei is a hyperthermophilic archaea and produces a heat stable polymerase (Pwo polymerase) that has proofreading activity.
Materials And Methods:
In this study, this microorganism was cultured, its DNA was extracted and the pwo gene polymerase was cloned, expressed and purified. The DNA sequence of the cloned gene was verified by sequencing. The pwo polymerase gene consists of 2,328 bps (775 amino acids with about 90 kD molecular weight). Cloning was done by GATEWAY™ Cloning System and for purification of recombinant protein; His6x-Tag was added to the C-terminus of the recombinant protein.
Results And Conclusion:
We could purify Pwo polymerase enzyme by Ni-NTA resin. PCR assay showed that Pwo polymerase activity is comparable to a commercial Pfu polymerase activity.
Insights
Researchers purified heat-stable Pyrococcus woesei DNA polymerase (Pwo polymerase) with proofreading activity. Its performance in PCR assays is comparable to commercial Pfu polymerase.
Area of Science:
- Biochemistry
- Molecular Biology
- Extremophile Research
Background:
- Pyrococcus woesei is a hyperthermophilic archaeon.
- It produces a heat-stable DNA polymerase with proofreading capabilities, known as Pwo polymerase.
Purpose of the Study:
- To clone, express, and purify the Pwo polymerase enzyme.
- To verify the DNA sequence of the cloned pwo gene.
- To assess the enzyme's activity and compare it to commercial alternatives.
Main Methods:
- Culturing of Pyrococcus woesei and DNA extraction.
- Cloning of the pwo gene using the GATEWAY™ Cloning System.
- Expression and purification of the recombinant protein with a C-terminal His6x-Tag using Ni-NTA resin.
- DNA sequencing to verify the gene sequence (2,328 bp, encoding 775 amino acids, ~90 kD).
Main Results:
- Successful purification of the Pwo polymerase enzyme.
- DNA sequencing confirmed the integrity of the cloned pwo gene.
- PCR assays demonstrated that Pwo polymerase activity is comparable to commercial Pfu polymerase.
Conclusions:
- The Pwo polymerase enzyme was successfully purified.
- The purified enzyme exhibits comparable PCR activity to Pfu polymerase, highlighting its potential utility.
