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A temperature-regulated replicon-based DNA expression system

Nature Biotechnology volume 18, pages 429–432 (2000)Cite this article

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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Figure 1: The CytTS expression vector carries the genes for the noncytopathic, temperature-sensitive viral replicase and the gene of interest (GOI) under the control of the subgenomic promoter (sg) and is stably integrated in the host cell chromosome.
Figure 2: Temperature dependence and levels of expression.
Figure 3: Kinetics of marker protein expression.
Figure 4: Production of β-IFN and EPO by the temperature-regulated expression system.
Figure 5: Generation of stable cell lines that express a toxic protein upon induction.

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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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  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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  1. Marco Boorsma
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  2. Lars Nieba
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Correspondence to Wolfgang A. Renner.

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Boorsma, M., Nieba, L., Koller, D. et al. A temperature-regulated replicon-based DNA expression system. Nat Biotechnol 18, 429–432 (2000). https://doi.org/10.1038/74493

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