Design of transcriptional fusions of stress sensitive promoters and GFP to monitor the overburden of Escherichia coli hosts during recombinant protein production.
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.
Nemecek
Sabine
S
Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
Marisch
Karoline
K
Juric
Renata
R
Bayer
Karl
K
D004926:Escherichia coli / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014158:Transcription, Genetic
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins