title: Plasmid-free T7-based Escherichia coli expression systems.
abstract: In order to release host cells from plasmid-mediated increases in metabolic load and high gene dosages, we developed a plasmid-free, T7-based E. coli expression system in which the target gene is site-specifically integrated into the genome of the host. With this system, plasmid-loss, a source of instability for conventional expression systems, was eliminated. At the same time, system leakiness, a challenging problem with recombinant systems, was minimized. The efficiency of the T7 RNA polymerase compensates for low gene dosage and provides high rates of recombinant gene expression without fatal consequences to host metabolism. Relative to conventional pET systems, this system permits improved process stability and increases the host cell's capacity for recombinant gene expression, resulting in higher product yields. The stability of the plasmid-free system was proven in chemostat cultivation for 40 generations in a non-induced and for 10 generations in a fully induced state. For this reason plasmid-free systems benefit the development of continuous production processes with E. coli. However, time and effort of the more complex cloning procedure have to be considered in relation to the advantages of plasmid-free systems in upstream-processing.
authors.affiliation: Austrian Center of Biopharmaceutical Technology, Muthgasse 18, A-1190 Vienna, Austria. gerald.striedner@boku.ac.at
authors.forename: Gerald
authors.initials: G
authors.lastname: Striedner
authors.forename: Irene
authors.initials: I
authors.lastname: Pfaffenzeller
authors.forename: Luchner
authors.initials: L
authors.lastname: Markus
authors.forename: Sabine
authors.initials: S
authors.lastname: Nemecek
authors.forename: Reingard
authors.initials: R
authors.lastname: Grabherr
authors.forename: Karl
authors.initials: K
authors.lastname: Bayer
mesh_terms: D017123:Bacteriophage T7 / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases