Overproduction of transcription termination factor Rho in Escherichia coli

Abstract

A plasmid system has been constructed which allows high-level expression of the rho gene of Escherichia coli under the control of the p L promoter and the N-antitermination regulatory system of bacteriophage A. The p L-directed synthesis of Rho crucially depends on the λ 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 p L 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.