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A temperature-regulated replicon-based DNA expression system

Nature Biotechnologyvolume 18pages429432 (2000) | Download Citation

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Abstract

We present a temperature-regulated, alphavirus replicon-based DNA expression system. The system is regulated by a viral temperature-sensitive RNA-dependent RNA replicase, creating a temperature-dependent RNA amplification loop. Because of this positive feedback, the system exhibits both low background and high inducibility. We observed 700-fold induction in transiently transfected cells, and over 104-fold induction in stably transfected cells. The high stringency of inducibility allowed the generation of stable cell lines expressing a highly toxic protein upon temperature shift. These data suggest that the present expression system could simplify bioprocess engineering strategies, especially in situations where the cloned protein has detrimental effects on host cell metabolism.

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Acknowledgements

We thank Prof. Sondra Schlesinger, Washington University (St. Louis, MO), for many helpful discussions and for providing the plasmids, p987BBneo and p987SinRep96. We acknowledge Dr. W.P.C. Stemmer, Maxygen (Redwood City, CA) for the GFP gene, Dr. P. Vito, Basel Institute for Immunology) for the RipDD gene, and we thank Drs. F. Hennecke, W. Hazenberg, and G. Orberger for helpful discussions.

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Affiliations

  1. Cytos Biotechnology AG, Wagistrasse 21, Zürich-Schlieren, CH-8952, Switzerland

    • Marco Boorsma
    • , Lars Nieba
    • , Daniel Koller
    • , Martin F. Bachmann
    •  & Wolfgang A. Renner
  2. Institute for Biotechnology, ETH-Zurich, CH-8093, Zurich, Switzerland

    • Marco Boorsma
    • , Daniel Koller
    •  & James E. Bailey

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Correspondence to Wolfgang A. Renner.

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https://doi.org/10.1038/74493

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