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.
Due to the lack of appropriate sensors for monitoring changes of Escherichia coli cells and the huge complexity of cellular systems, many of the present protein production processes are still far from optimal. Aiming at maximal exploitation of the host cell, enhanced knowledge of cellular reactions related to recombinant protein expression is required. Current methods like DNA microarrays and 2-D-electrophoresis enable the acquisition of transcriptional and translational activity shifts in stress situations like heat shock, general stress response, nutrient limitation, and stress caused by overexpression of heterologous proteins. However, these techniques and data processing are time consuming, therefore, the goal is to create new on-line systems such as stress promoter GFP fusions to monitor metabolic alterations. The fluorescence signal of expressed GFP can be measured by 2-D-multi-wavelength fluorescence spectroscopy, thereby allowing non-invasive on-line in vivo monitoring. Results of efficient stress monitoring approaches in ongoing protein production process are presented.
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