Modified bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli

Abstract

A new series of expression vectors that direct high-level overproduction of gene products in Escherichia coli is described. All contain strong bacteriophage lambda promoters, PR and PL, arranged in tandem so that both promote transcription into genes inserted into or between unique restriction sites. The vectors also direct expression of the lambda cI857 gene (from its natural promoter, PM), which enables their use in any E. coli host strain to effect controlled expression by shifting the temperature of cultures from 30 to 42 degrees C. The vectors pCE30, pND201, pPT150 and pMA200U are derivatives of the high-copy-number plasmid pUC9. Vector pCE33 is an analogous derivative of the heat-inducible runaway-replication plasmid, pMOB45, and directs overproduction of proteins by virtue of increase in both gene dosage and transcription following treatment at 42 degrees C. The vectors pND201 and pPT150 bear a ribosome-binding site (RBS) perfectly complementary to the 3' end of E. coli 16-S rRNA a few bp upstream from a unique HpaI site. Ways in which they may be used to improve the efficiency of translation of mRNA by substitution of a natural RBS with selection for optimal spacing from an ATG (or GTG) start codon are described. The phagemid vector pMA200U is a direct analog of pCE30 designed to facilitate preparation of single-stranded DNA templates for use in oligodeoxyribonucleotide-directed mutagenesis of overexpressed genes.