Plasmid‐free T7‐based Escherichia coli expression systems
- 4 November 2009
- journal article
- research article
- Published by Wiley in Biotechnology & Bioengineering
- Vol. 105 (4), 786-794
- https://doi.org/10.1002/bit.22598
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. Biotechnol. Bioeng. 2010. 105: 786–794.Keywords
This publication has 43 references indexed in Scilit:
- Npro fusion technology to produce proteins with authentic N termini in E. coliNature Methods, 2007
- Plasmid Copy Number and Plasmid StabilityAdvances in Biochemical Engineering/biotechnology, 2003
- One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR productsProceedings of the National Academy of Sciences, 2000
- Plasmid Effects onEscherichia coliMetabolismCritical Reviews in Biotechnology, 2000
- Metabolic approaches for the optimisation of recombinant fermentation processesApplied Microbiology and Biotechnology, 1999
- Recombinant protein expression in Escherichia coliCurrent Opinion in Biotechnology, 1999
- FACS-optimized mutants of the green fluorescent protein (GFP)Gene, 1996
- Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressorJournal of Molecular Biology, 1991
- Plasmid‐encoded protein: The principal factor in the “metabolic burden” associated with recombinant bacteriaBiotechnology & Bioengineering, 1990
- Studies of Host‐Plasmid Interactions in Recombinant MicroorganismsaAnnals of the New York Academy of Sciences, 1986