title: The use of elements of the E. coli Ntr-system for the design of an optimized recombinant expression system for high cell density cultivations.
abstract: The inducible glnA promoter 2 of the E. coli glutamine synthetase gene is suitable as an expression unit for the production of recombinant proteins at low and high cell densities. It is active when the concentration of ammonium as the sole nitrogen source in the culture medium is below 1 mM. This nitrogen regulatory system was optimized by introduction of expression cassettes consisting of additional elements of the ntr-system. These artificial constructions result in enhanced recombinant gene expression in the production phase. Furthermore, the basic recombinant protein level during the growth phase is reduced due to a tighter promoter control. A three- to four-fold higher accumulation of chloramphenicol-acetyltransferase (as reporter protein) and of anti-EGF-receptor miniantibodies was achieved by increasing the amount of the final regulator molecule NtrC approximately P via plasmidal co-expression of the ntrC gene. The introduction of a modified glnA promoter 1 inverse to glnAp2 lowered the basic activity of glnAp2 to about one half. It is assumed that under nitrogen excess conditions sigma 70-RNA polymerase binds at glnAp1 and thereby prevents most of the binding of sigma 54-RNA polymerase at glnAp2. The optimized expression systems were successfully applied in low and high cell density cultivations. In the fed-batch phase of high cell density cultivations recombinant protein formation was induced through external nitrogen limitation under FIA-controlled concentration of glucose as carbon source.
authors.affiliation: Hans Knöll Institute for Natural Products Research, Jena, Germany. schroeck@pmail.hki-jena.de
authors.forename: V
authors.initials: V
authors.lastname: Schroeckh
authors.forename: R
authors.initials: R
authors.lastname: Wenderoth
authors.forename: M
authors.initials: M
authors.lastname: Kujau
authors.forename: U
authors.initials: U
authors.lastname: Knüpfer
authors.forename: D
authors.initials: D
authors.lastname: Riesenberg
mesh_terms: D000906:Antibodies / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002455:Cell Division / Q000235:genetics; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism; D051922:PII Nitrogen Regulatory Proteins; D011401:Promoter Regions, Genetic; D051877:RNA Polymerase Sigma 54; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*; D012808:Sigma Factor / Q000378:metabolism; D015534:Trans-Activators; D014157:Transcription Factors
chemical_list: D000906:Antibodies; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D051922:PII Nitrogen Regulatory Proteins; D011994:Recombinant Proteins; D012808:Sigma Factor; D015534:Trans-Activators; D014157:Transcription Factors; C497876:glnG protein, E coli; C497751:rpoN protein, E coli; D004815:Epidermal Growth Factor; D015500:Chloramphenicol O-Acetyltransferase; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; C081173:glutamine synthetase I; D005974:Glutamate-Ammonia Ligase; D009584:Nitrogen