Cloning, expression and purification of Pwo polymerase from Pyrococcus woesei
Ghasemi، Amir نويسنده Associate Professor, Operative Dentistry, School of Dentistry, Shahid Behesht University of Medical Science. Shahid Behesht, Iran , , Hatef Salmanian، Ali نويسنده National Institute of Genetic Engineering and Biotechnology, Tehran, IR Iran , , Sadeghifard، Nourkhoda نويسنده Department of Microbiology, Faculty of Medicine, Ilam University of Medical Sciences, Ilam, Iran Sadeghifard, Nourkhoda , Salarian، Amir-Ahmad نويسنده Army University of Medical Sciences, Tehran, Iran. Salarian, Amir-Ahmad , Khalifeh Gholi، Mohammad نويسنده Department of Pathobiology, Institute of Public Health, Tehran University of Medical Sciences, Tehran, Iran. Khalifeh Gholi, Mohammad
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.