A new cloning vector and expression strategy for genes encoding proteins toxic to Escherichia coli.
Here, we describe a modification of a plasmid, pT7-7 [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 262 (1985) 1074-1078], that allows expression of inserted genes from the phage T7 RNA polymerase promoter. The modification is designed to suppress readthrough transcription from cryptic promoters and start points on the plasmid, in order to reduce expression in the absence of T7 RNA polymerase and thus improve the vector for use in the expression of highly toxic gene products. This vector (pT7SC) was used to stably clone the POL3 gene (encoding DNA polymerase delta) of Saccharomyces cerevisiae, which destabilizes all other cloning and expression vectors tested. Previously described expression strategies proved ineffective in overexpressing the POL3 gene. A new strategy was developed which relies on induction by infection with mutant T7 phage. This system efficiently overproduced the POL3 gene product.
Brown
W C
WC
Braun Laboratories, Division of Chemistry, California Institute of Technology, Pasadena 91125.
Campbell
J L
JL
D017123:Bacteriophage T7 / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D003603:Cytotoxins / Q000235:genetics*; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005822:Genetic Vectors; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017384:Sequence Deletion; D014158:Transcription, Genetic
D003603:Cytotoxins; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase