LOW-COPY-NUMBER T7 VECTORS FOR SELECTIVE GENE-EXPRESSION AND EFFICIENT PROTEIN OVERPRODUCTION IN ESCHERICHIA-COLI

A set of low-copy-number vectors (pPD) has been constructed that permi t selective gene expression and high-level protein overproduction in E scherichia coli, based on the bacteriophage T7 RNA polymerase/T7 promo ter system. These plasmids carry a chloramphenicol resistance gene (ca t) as a selective marker and an extended multiple cloning site for con venient gene cloning. Their replication is mediated by ori sequences d erived from the low-copy-number vector pSC101. The efficient T7 gene 1 0 promoter present on these vectors allows selective and high-level tr anscription of cloned genes carrying their own translational initiatio n signals. In addition, low-copy-number T7 vectors were constructed th at permit expression of genes lacking their own transcription and tran slation initiation elements by providing a ribosome binding site, an A TG start codon and a multiple cloning site devised for the cloning in all three reading frames. The pPD expression vectors were used to achi eve high-level overproduction of the E. coli integral outer membrane p rotein Tsx, and the cytoplasmic enzymes beta-galactosidase (beta Gal) and UTP:alpha-D-glucose-1-phosphate uridylyltransferase (GalU). The ch aracteristics of these low-copy-number T7 expression vectors should pr ove very useful for the cloning and high-level overexpression of genes whose gene products are deleterious to the E. coli host.