title: Tight transcriptional control mechanism ensures stable high-level expression from T7 promoter-based expression plasmids.
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 lambda-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.
authors.affiliation: Laboratory of Molecular Biology, University of Gent, Belgium. nico@lmb1.rug.ac.be
authors.forename: N
authors.initials: N
authors.lastname: Mertens
authors.forename: E
authors.initials: E
authors.lastname: Remaut
authors.forename: W
authors.initials: W
authors.lastname: Fiers
mesh_terms: D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics*; D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics; D014764:Viral Proteins
chemical_list: D016207:Cytokines; D014409:Tumor Necrosis Factor-alpha; D014764:Viral Proteins; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases