TIGHT TRANSCRIPTIONAL CONTROL MECHANISM ENSURES STABLE HIGH-LEVEL EXPRESSION FROM T7 PROMOTER-BASED EXPRESSION PLASMIDS

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 spe cific T7 RNA polymerase. Using various T7 RNA polymerase/T7 promoter-b ased vector host systems with differential control on expression of th e T7 RNA polymerase,,ve document that leaky expression of the latter i s responsible for the frequently observed loss of the culture's abilit y to express genes of interest. We further show that the inability to achieve detectable expression levels can be overcome by using a tightl y repressed expression system. We describe a novel and efficient contr ol system in which basal level expression of T7 RNA polymerase is atte nuated by a series of tandemly arranged transcription terminators. The plasmids also incorporate the phage lambda-derived nutL/N protein ant itermination function, allowing conditional reversion of attenuation u pon induction. The applicability of the system is illustrated by the s trictly regulatable, high-level production of several cytokines of hum an and murine origin.