title: Use of a modified tryptophanase promoter to direct high-level expression of foreign proteins in E. coli.
abstract: We have modified the tryptophanase promoter (PtnaA) for use as a temperature-independent promoter for the production of recombinant proteins. Although any protein will have a temperature range in which its expression is optimal, we find the tryptophanase promoter functions at all physiologically relevant temperatures (20 degrees C to 42 degrees C). Induction at temperatures below 37 degrees C avoids eliciting the heat-shock response and may favor the production of protein in the soluble state. A short segment of the E. coli tnaA promoter containing the catabolite gene activator protein (CAP) binding site but no tryptophan-responsive elements was used to direct synthesis of various proteins. Conditions for high cell density fermentation and induction control were developed. Expression was induced by depletion of glucose and was maximal when an alternative nonrepressing carbon source was supplied. Expression of certain proteins was tightly controlled; however, pre-induction expression was observed with other reporter genes. The tnaC leader portion of the tnaA promoter was found to reduce pre-induction expression in the presence of glucose, although maximal expression was observed only in the absence of this region. The effect of temperature on expression of several recombinant proteins was investigated. Although some proteins were produced only in inclusion bodies as insoluble material, the production of one protein in soluble form was clearly temperature dependent.
authors.affiliation: Microbiology and Applied Microbial Genetics, Amgen Inc., Amgen Center, Thousand Oaks, California 91320, USA.
authors.forename: K C
authors.initials: KC
authors.lastname: Sitney
authors.forename: M B
authors.initials: MB
authors.lastname: Mann
authors.forename: G W
authors.initials: GW
authors.lastname: Stearns
authors.forename: A D
authors.initials: AD
authors.lastname: Menjares
authors.forename: J L
authors.initials: JL
authors.lastname: Stevenson
authors.forename: M D
authors.initials: MD
authors.lastname: Snavely
authors.forename: J C
authors.initials: JC
authors.lastname: Fieschko
authors.forename: C
authors.initials: C
authors.lastname: Curless
authors.forename: L B
authors.initials: LB
authors.lastname: Tsai
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods*; D002373:Cyclic AMP Receptor Protein / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005285:Fermentation; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D015183:Restriction Mapping; D017384:Sequence Deletion; D013696:Temperature; D014368:Tryptophanase / Q000235:genetics*
chemical_list: D002373:Cyclic AMP Receptor Protein; D011994:Recombinant Proteins; D014368:Tryptophanase; D005947:Glucose