Simultaneous regulation of plasmid replication and heterologous gene expression in Escherichia coli.
An expression plasmid in which plasmid DNA replication and heterologous gene expression can be simultaneously regulated was constructed to avoid derepression prior to induction. This was achieved by placing a pBR322 origin of replication immediately downstream of an anthranilate synthase-human epidermal growth factor fusion gene (trpE-hEGF), both under the control of the promoter from the tryptophan biosynthetic operon. Regulation of plasmid copy number ensured tight repression of the trp promoter prior to induction. Upon induction, plasmid copy number increased up to six-fold and the fusion protein accumulated to approximately 12% of total cell protein. Induction experiments with a series of plasmid derivatives with sequentially lower copy numbers revealed that accumulation levels of the TrpE-hEGF fusion protein post-induction correlated well with plasmid copy number. Plasmid constructs where the native trp promoter had been replaced by derivatives deleted of the attenuator resulted in high levels of hEGF accumulation in the tryptophan-free medium prior to induction. Nevertheless, up to two-fold increase in TrpE-hEGF accumulation levels were obtained using the constructs lacking the attenuator compared to those bearing the native trp promoter.
Department of Biological Sciences, Monsanto Company, St. Louis, Missouri 63198.
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D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014364:Tryptophan / Q000235:genetics
D004269:DNA, Bacterial; D004815:Epidermal Growth Factor; D014364:Tryptophan