Summary
We have investigated the effect of alterations in the structure of the plasmid-borne Escherichia coli tryptophan (trp) coding region and other regions of the same replicon on the level, rate and time of initiation of anthranilate synthetase component I (ASase) synthesis in E. coli K12. The maximum level of ASase produced corresponds to 60%–65% of the total cellular proteins. Adding sequences downstream of the trpE coding region decreases the level but does not affect the time of initiation and rate of trpE expression (ASase synthesis). The presence of additional protein coding sequences on the plasmid outside the trpE-A region causes ASase production to start earlier and decreases the rate of ASase synthesis. A second copy of the trpE coding sequences, if present within or outside the trpE-A coding region on the same replicon, doubles the rate of synthesis of ASase and slightly increases its final level of production. The initiation of ASase production occurs earlier when the two trpE copies are located within two distinct transcription units.
Similar content being viewed by others
References
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 72:248–254
Broekhuijsen MP, Blom T, Van Rijn J, Pouwels PH, Klasen EA, Fasbender MJ, Enger-Valk BE (1986) Synthesis of fusion proteins with multiple copies of an antigenic determinant of foot-and-mouth disease virus. Gene 49:189–197
Clarke L, Carbon J (1978) Functional expression of cloned yeast DNA in E. coli: specific complementation of arginino-succinate lyase (argH) mutations. J Mol Biol 120:517–532
Cohen G, Jacob F (1959) Sur la répression de la synthése des enzymes intervenant dans la formation du tryptophane chez E. coli. CR Acad Sci (Paris) 248:3490–3492
Doolittle WF, Yanofsky C (1968) Mutants of Escherichia coli with an altered tryptophanyl-transfer ribonucleic acid synthetase. J Bacteriol 95:1283–1294
Enger-Valk BE, Heyneker HL, Oosterbaan RA, Pouwels PH (1980) Construction of new cloning vehicles with genes of the tryptophan operon of Escherichia coli as genetic markers. Gene 9:69–85
Gibson F (1970) Preparation of chorismic acid. Methods Enzymol 17:362–364
Goeddel DV, Heyneker HL, Hozumi T, Arentzen R, Itakura K, Yansura DG, Ross MJ, Miozarri G, Crea R, Seeburg PS (1979) Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone. Nature 281:544–548
Guyer MS, Reed RR, Steitz JA, Low LB (1981) Identification of a Sex-factor-affinity Site in E. coli as γδ. Cold Spring Harbor Symp Quant Biol 45:135–140
Ito J, Cox EC, Yanofsky C (1969) Anthranilate Synthetase, an enzyme specified by the tryptophan operon of Escherichia coli: purification and characterization of Component I. J Bacteriol 97:725–733
Jay E, Rommens J, Pomeroy-Cloney L, MacKnight D, Lutze-Wallace C, Wishart P, Harrison D, Lui WY, Asundi V, Dawood M, Jay F (1984) High-level expression of a chemically synthesized gene for human interferon-γ using a prokaryotic expression vector. Proc Natl Acad Sci USA 81:2290–2294
Kos A, Boekhuijsen MP, Van Gorcom R, Van Rotterdam J, Enger-Valk BE, Veltkamp E, Van den Hondel C, Pouwels PH (1984) Construction of vectors suitable for efficient expression of genes in E. coli. In: Houwink EH, Van der Meer RR (eds) Innovations in biotechnology. Elsevier, Amsterdam, pp 415–425
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680–685
Lee N, Cozzitorto J, Wainwright N, Testa D (1984) Cloning with tandem gene systems for high level gene expression. Nucleic Acids Res 12:6797–6812
Malloy JM, Rieker JP, Rizzo CF (1984) Quantitation of proteins on Coomassie Blue-stained polyacrylamide gels based on spectrophotometric determination of electroeluted dye. Anal Biochem 141:503–509
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Rose JK, Squires CL, Yanofsky C, Yang H-L, Zubay G (1973) Regulation of in vitro transcription of the tryptophan operon by purified RNA polymerase in the presence of partially purified repressor and tryptophan. Nature New Biol 245:133–137
Saito T, Watanabe Y, Meshi T, Okada Y (1986) Preparation of antibodies that react with the large non-structural proteins of tobacco mosaic virus by using Escherichia coli expressed fragments. Mol Gen Genet 201:82–89
Author information
Authors and Affiliations
Additional information
Communicated by J. Schell
Rights and permissions
About this article
Cite this article
Hessing, H.G.M., van Rotterdam, C. & Pouwels, P.H. Expression of the Escherichia coli trpE gene in E. coli K12 bacteria: Maximum level, rate and time of initiation of anthranilate synthetase production. Molec Gen Genet 210, 256–261 (1987). https://doi.org/10.1007/BF00325691
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00325691