Gene expression from multicopy T7 promoter vectors proceeds at single copy rates in the absence of T7 RNA polymerase. 
Three different genes (trpR+, tyrR+ and phi (trpR-lacZ)) were inserted into pET3a, a multicopy transcription-translation vector designed by Rosenberg et al. (1) for the T7 RNA polymerase-driven overexpression of proteins in Escherichia coli. Gene orientation was in the anticlockwise (&quot;silent&quot;) direction. Gene expression in the absence of T7 RNA polymerase was evaluated either directly using lacZ reporter systems or indirectly by observing the susceptibility of plasmid-bearing tester strains to inhibition by an aromatic amino acid analog. The production of repressor proteins and of a Trp repressor-LacZ chimera was readily detected, at levels comparable to those of haploid trpR+ or tyrR+ E. coli strains. Such T7 vector constructs thus have two especially useful properties: first, they provide a means for the high-level production of various proteins in E. coli; second, they offer a technically advantageous point of departure for structure-function studies of genes whose overexpression from multicopy plasmids would normally be cytotoxic. 