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Overexpression of a lethal methylase, M.TneDI, in E. coli BL21(DE3)

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An Erratum to this article was published on 17 June 2014

Abstract

A pET-based vector pDH21 expressing the methylase, M.TneDI (recognizing CGCG) from Thermotoga was constructed, and transformed into E. coli BL21(DE3). Despite E. coli BL21(DE3) being McrBC positive, 30 transformants were isolated, which were suspected to be McrBC− mutants. The overexpression of M.TneDI was verified by SDS-PAGE analysis. Compared to the previously constructed pJC340 vector, a pACYC184 derivative expressing M.TneDI from a tet promotor, the newly constructed pDH21 vector improved the expression of the methylase about fourfold, allowing complete protection of DNA substrates. This study not only demonstrates a practical approach to overexpressing potential lethal proteins in E. coli but also delivers a production strain of M.TneDI that may be useful in various in vitro methylation applications.

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Acknowledgments

We are grateful to Dr. Robert M. Blumenthal at the University of Toledo, College of Medicine for sharing plasmid pPvuM1.9. This work was supported by the BGSU Commercialization Catalyst Award and the Building Strength Award.

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Authors and Affiliations

  1. Department of Biological Sciences, Bowling Green State University, Bowling Green, OH, 43403, USA

    Hui Xu, Dongmei Han & Zhaohui Xu

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  1. Hui Xu
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  2. Dongmei Han
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  3. Zhaohui Xu
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Correspondence to Zhaohui Xu.

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Xu, H., Han, D. & Xu, Z. Overexpression of a lethal methylase, M.TneDI, in E. coli BL21(DE3). Biotechnol Lett 36, 1853–1859 (2014). https://doi.org/10.1007/s10529-014-1552-z

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  • DOI: https://doi.org/10.1007/s10529-014-1552-z

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