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 ("silent") 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.
Somerville
R L
RL
Department of Biochemistry, Purdue University West Lafayette, IN 47907.
Shieh
T L
TL
Hagewood
B
B
Cui
J S
JS
D001426:Bacterial Proteins / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics*; D015183:Restriction Mapping; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics; D014364:Tryptophan / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; C027190:TRPR protein, E coli; D014157:Transcription Factors; C069256:TyrR protein, E coli; D014364:Tryptophan; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase