University of Natural Resources and Life Sciences, Vienna (BOKU) - Research portal
Gewählte Master / Diploma Thesis:
Karin Duerrschmid
(2000):
Anwendung der 2D-Elektrophorese zur Untersuchung der Proteinexpression rekombinanter Escherichia coli.
Master / Diploma Thesis,
BOKU-Universität für Bodenkultur.
UB BOKU
obvsg
- Abstract:
- Monitoring of the metabolic burden on the host cell metabolism using stress response signals is an important issue for the optimisation of recombinant fermentation processes. The main goal of this work was the investigation of the impact of recombinant protein synthesis on the composition of cellular proteins using two dimensional electrophoresis (2D-E). The aim was to find marker proteins, which enable qualitative and quantitative evaluation of the metabolic burden on host cell. To be able to monitor shifts in the protein expression program due to re- combinant protein production 2D-E had to be further optimised. Crucial parameters are resolution power and reproducibility. Optimal performance was attained through variation of focussing times, gels and selection of staining methods. Although differences of the protein maps showed signifi- cant variation during recombinant protein expression specific stress rela- ted marker proteins could not be entirely identified. However, the dis- appearance of the spot of the glucose specific IIA component of the phos- photransferase system can be assumed as a specific signal. This protein is phosphorylated due to glucose limitation and triggers the synthesis of cyclic AMP, a signal of stress response. Hence it becomes part of the stress regulatory mechanisms. The changes of the protein pattern provide evidence of the differentiation of the cell caused by the metabolic burden. The impact of this differentiation process on recombinant protein produc- tion has to be investigated more in depth. In that way recently developed gene constructs provide valuable tools to receive important information on the complex interaction between recombinant protein expression and host cell metabolism.
- Beurteilende(r): Bayer Karl