Cloning, expression and purification of Pwo polymerase from Pyrococcus woesei

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, expr...

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Autores principales: Ghasemi, Amir, Salmanian, Ali Hatef, Sadeghifard, Nourkhoda, Salarian, Amir Ahmad, Gholi, Mohammad Khalifeh
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Tehran University of Medical Sciences 2011
Materias:
Original Artical
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3279813/
https://www.ncbi.nlm.nih.gov/pubmed/22347593
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author Ghasemi, Amir
Salmanian, Ali Hatef
Sadeghifard, Nourkhoda
Salarian, Amir Ahmad
Gholi, Mohammad Khalifeh
author_facet Ghasemi, Amir
Salmanian, Ali Hatef
Sadeghifard, Nourkhoda
Salarian, Amir Ahmad
Gholi, Mohammad Khalifeh
author_sort Ghasemi, Amir
collection PubMed
description 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.
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spelling pubmed-32798132012-02-16 Cloning, expression and purification of Pwo polymerase from Pyrococcus woesei Ghasemi, Amir Salmanian, Ali Hatef Sadeghifard, Nourkhoda Salarian, Amir Ahmad Gholi, Mohammad Khalifeh Iran J Microbiol Original Artical 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. Tehran University of Medical Sciences 2011-09 /pmc/articles/PMC3279813/ /pubmed/22347593 Text en © 2011 Iranian Society of Microbiology & Tehran University of Medical Sciences http://creativecommons.org/licenses/by/3.0/ This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.
spellingShingle Original Artical
Ghasemi, Amir
Salmanian, Ali Hatef
Sadeghifard, Nourkhoda
Salarian, Amir Ahmad
Gholi, Mohammad Khalifeh
Cloning, expression and purification of Pwo polymerase from Pyrococcus woesei
title Cloning, expression and purification of Pwo polymerase from Pyrococcus woesei
title_full Cloning, expression and purification of Pwo polymerase from Pyrococcus woesei
title_fullStr Cloning, expression and purification of Pwo polymerase from Pyrococcus woesei
title_full_unstemmed Cloning, expression and purification of Pwo polymerase from Pyrococcus woesei
title_short Cloning, expression and purification of Pwo polymerase from Pyrococcus woesei
title_sort cloning, expression and purification of pwo polymerase from pyrococcus woesei
topic Original Artical
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3279813/
https://www.ncbi.nlm.nih.gov/pubmed/22347593
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