Construction of an overproduction vector containing the novel srp (sterically repressed) promoter.
We report the design, synthesis, and evaluation of a novel Escherichia coli promoter intended for use in overproduction of proteins that are deleterious to the host. In this sterically repressed promoter (srp), the lac operator site is positioned between the -10 and -35 elements, where it can interfere sterically with RNA polymerase and thereby prevent assembly of a poised transcriptional complex. An srp-containing phagemid, pKEN1, and a tac-containing phagemid, pHN1, which has been widely used in protein overproduction but is often unstable, are compared with respect to levels of uninduced and induced protein expression. The level of uninduced protein synthesis by the srp promoter in vivo is approximately 50% of that observed with tac, whereas the levels of induced protein synthesis with the 2 vectors are approximately equal. A remarkable increase in stability of overproduction and growth was observed when the toxic Ada protein was overproduced in pKEN1, demonstrating the potential utility of this vector in overproducing toxic proteins.
Ezaz-Nikpay
K
K
Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138.
Uchino
K
K
Lerner
R E
RE
Verdine
G L
GL
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001435:Bacteriophages; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D007763:Lac Operon; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D015183:Restriction Mapping; D014157:Transcription Factors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D014157:Transcription Factors; C045675:Ada protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase; D012321:DNA-Directed RNA Polymerases