Cloning, expression and purification of Pwo polymerase from Pyrococcus woesei

Pyrococcus woesei is a hyperthermophilic archaea and produces a heat stable polymerase [Pwo polymerase] that has proofreading activity. 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. 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