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Tight Transcriptional Control Mechanism Ensures Stable High-Level Expression from T7 Promoter-Based Expression Plasmids

Bio/Technology volume 13, pages 175–179 (1995)Cite this article

Abstract

One of the more efficient systems for high-level expression of cloned genes in Escherichia coli makes use of a phage T7 late promoter whose activity depends on a regulatable transcription unit supplying the specific T7 RNA polymerase. Using various T7 RNA polymerase/T7 promoter-based vector host systems with differential control on expression of the T7 RNA polymerase, we document that leaky expression of the latter is responsible for the frequently observed loss of the culture's ability to express genes of interest. We further show that the inability to achieve detectable expression levels can be overcome by using a tightly repressed expression system. We describe a novel and efficient control system in which basal level expression of T7 RNA polymerase is attenuated by a series of tandemly arranged transcription terminators. The plasmids also incorporate the phage λ-derived nutL / N protein antitermination function, allowing conditional reversion of attenuation upon induction. The applicability of the system is illustrated by the strictly regulatable, high-level production of several cytokines of human and murine origin.

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Author notes

  1. Walter Fiers: e-mail: nico@lmbl.rug.ac.be

Authors and Affiliations

  1. Laboratory of Molecular Biology, University of Gent, K. L. Ledeganckstraat 35, B-9000, Gent, Belgium

    Nico Mertens, Erik Remaut & Walter Fiers

Authors
  1. Nico Mertens
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  2. Erik Remaut
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  3. Walter Fiers
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Mertens, N., Remaut, E. & Fiers, W. Tight Transcriptional Control Mechanism Ensures Stable High-Level Expression from T7 Promoter-Based Expression Plasmids. Nat Biotechnol 13, 175–179 (1995). https://doi.org/10.1038/nbt0295-175

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