A novel phosphate-regulated expression vector in Escherichia coli.
The ugp promoter (pugp) responsible for expression of the binding-protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli was cloned into a small multicopy plasmid pTER5, a derivative of pBR322, between the transcription terminators rpoCt and tL1. The resulting expression vector, pPH3, permits convenient insertion of structural genes containing their own translational-initiation regions, into the multiple-cloning site derived from the pUC19 plasmid. The efficiency and regulatory properties of pugp were measured using xylE and lacZ as reporter genes, which code for the corresponding enzymes catechol-2,3-dioxygenase (C23O) and beta-galactosidase (beta Gal), respectively. Enzyme activities were virtually completely repressed in the presence of excess inorganic phosphates (Pi) and high concentrations of glucose. Maximal induction was observed at limiting Pi (less than 0.1 mM) and normal levels of glucose (0.2-0.4%). The maximum expression of the pugp-directed beta Gal synthesis was approx. 80% of that directed by strong ptac. When the xylE gene was maximally expressed, the induced enzyme constituted approx. 50% of total cellular protein as judged by laser densitometry following sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. These results suggest the usefulness of the pugp in expression vectors for strong, but controlled, expression of cloned genes in E. coli. This Pi controlled vector can be adapted to large-scale fermentation by using Pi-limiting growth conditions.
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Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
Schweizer
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Oxender
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D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005822:Genetic Vectors; D005947:Glucose / Q000494:pharmacology; D005994:Glycerophosphates / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology*; D010957:Plasmids; D011401:Promoter Regions, Genetic
D005994:Glycerophosphates; D010710:Phosphates; D005947:Glucose