Overproduction of transcription termination factor Rho in Escherichia coli.
A plasmid system has been constructed which allows high-level expression of the rho gene of Escherichia coli under the control of the pL promoter and the N-antitermination regulatory system of bacteriophage lambda. The pL-directed synthesis of Rho crucially depends on the lambda N gene product and is promoted most effectively when this product is supplied from the N gene cloned on a separate compatible plasmid with a moderate copy number. The requirement for N can be circumvented partly, but not completely, by deletion of the region preceding the rho structural gene. Attempts were also made to optimize the construction of rho-expression plasmids by adjusting the orientation and location of pL and rho inserts on the pBR322 vector. With optimal conditions, Rho protein is overexpressed 100-fold and can become as much as 10% of the total cellular protein. Using this plasmid system, Rho can be purified with a yield of more than 20 mg from 10 g of induced cells.
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D009876:Operon; D010957:Plasmids; D012234:Rho Factor / Q000096:biosynthesis / Q000235:genetics*; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics*
D012234:Rho Factor; D014157:Transcription Factors; D004262:DNA Restriction Enzymes