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Cloning, Expression and Purification of Pwo Polymerase from Pyrococcus Woesei

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Author(s): A Ghasemi | A Hatef Salmanian | S Sadeghifard | A Salarian | M Khalifeh Gholi

Journal: Iranian Journal of Microbiology
ISSN 2008-3289

Volume: 3;
Issue: 3;
Start page: 118;
Date: 2011;
Original page

Keywords: Pyrococcus Woesei | Cloning | PCR

ABSTRACT
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 GATEWAYTM 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.
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