Cloning, expression, and purification of the His(6)-tagged hyper-thermostable dUTPase from Pyrococcus woesei in Escherichia coli: application in PCR

Slawomir Dabrowski, Birgitte Kiær Ahring

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    The gene encoding dUTPase from Pyrococcus woesei was cloned into Escherichia coli expression system. It shows 100% gene identity to homologous gene in Pyrococcus furiosus. The expression of N-terminal His(6)-tagged Pwo dUTPase was performed in E coli BL21(DE3)pLysS and E. coli Rosetta(DE3)pLysS strain that contains plasmid encoding additional copies of rare E. coli tRNAs. E. coli Rosetta(pLysS) strain was found with two times higher expression yield of His(6)-tagged Pwo dUTPase than E. coli BL21(DE3)pLysS. The His(6)-tagged Pwo dUTPase was purified on Ni2+-IDA-Sepharose, dialyzed, and the enzyme activity was investigated. We found that His(6)-tag domain has no influence on dUTP hydrolytic activity. dUTP is generated during PCR from dCTP, which inhibits the polymerization of DNA catalyzed by DNA polymerase with 3'-5' exonuclease activity. We observed that the thermostable His(6)-tagged Pwo dUTPase used for the polymerase chain reaction with P. woesei DNA polymerase improves the efficiency of PCR and it allows for amplification of longer targets.
    Original languageEnglish
    JournalProtein Expression and Purification
    Volume31
    Issue number1
    Pages (from-to)72-78
    ISSN1046-5928
    Publication statusPublished - 2003

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