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

• Published in: Bio/Technology 1995-02, Vol.13 (2), p.175-179
• Main Authors: Mertens, Nico, Remaut, Erik, Fiers, Walter
• Format: Article
• Language: English
• Subjects: Agriculture; Animals; Bacteriophage lambda - genetics; Bacteriophage T7 - genetics; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Cloning, Molecular; Cytokines - biosynthesis; Cytokines - genetics; DNA - chemistry; DNA - metabolism; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation; Genetic engineering; Genetic technics; Humans; Life Sciences; Methods. Procedures. Technologies; Mice; Plasmids - genetics; Promoter Regions, Genetic; Transcription, Genetic; Tumor Necrosis Factor-alpha - genetics; Vectors (cloning, transfer, expression). Insertion sequences and transposons; Viral Proteins
• ISSN: 0733-222X; 1087-0156; 2331-3684; 1546-1696
• DOI: 10.1038/nbt0295-175

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 nut L / 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.