Team Leader (USP)

PD Dr. Yvonne Genzel
PD Dr. Yvonne Genzel
Phone: +49 391 6110 257
Room: N0.18

Researcher

Dipl.-Biol. (t.o.) Thomas Bissinger
Dipl.-Biol. (t.o.) Thomas Bissinger
Phone: +49 391 6110 131
Room: N 0.07

Additional Information

Collaborations:

MPI of Molecular Plant Physiology, Golm, System Regulation, Department of Metabolic Networks
(Prof. Dr. M. Stitt)

Enzymatic Characterization of Mammalian Cells

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Enzymatic Characterization of Mammalian Cells

Motivation

Mammalian cell cultures are commonly used for the production of recombinant proteins and vaccines. Thus, the knowledge of their metabolic capacity has great potential for process optimization by improving cultivation conditions as well as by designing cell lines using directed genetic modifications. In particular, platforms for the characterization of metabolome, fluxome, and snap shots of a given enzyme activity status/maximum enzyme activities can provide functional and regulatory knowledge about cell metabolism.

Aim of the project

To examine cell metabolism and regulatory interactions in more detail, the project focuses on the measurement of intracellular enzyme activities. Therefore, a high-throughput platform established for enzyme activity measurements in plant cells was first adapted to adherent mammalian cell lines and then further adapted to suspension cell lines (in cooperation with the MPI for Plant Physiology). Changes in enzyme levels are analyzed under different cultivation conditions, primarily focusing on glycolysis, glutaminolysis, citrate cycle, and pentose phosphate pathway. Enzyme activities are essential for the analysis of metabolic and regulatory networks, where disagreements between flux and metabolite levels hint to allosterically controlled enzymes and very low or missing activity levels indicate missing pathways.

For the determination of enzyme activities, cycling assays are used to achieve a very high sensitivity compared to direct enzyme activity measurements. Thus, cell extracts can be monitored at high dilution. Interfering side reactions and concentration of inhibiting substances are also reduced. Furthermore, only very small sample volumes are required, which allow high throughput small-scale cultivations.cale cultivations [1].

Fig. 1 Principle of stopped assays for the determination of enzyme activities. The product of the reaction is determined in the respective cycling assay. Here, NAD+-cycling. Zoom Image
Fig. 1 Principle of stopped assays for the determination of enzyme activities. The product of the reaction is determined in the respective cycling assay. Here, NAD+-cycling.
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References

Lowry, O. H.:
Amplification by enzymatic cycling.
Janke, R.; Genzel, Y.; Wahl, A.; Reichl, U.: Measurement of Key Metabolic Enzyme Activities in Mammalian Cells Using Rapid and Sensitive Microplate-Based Assays. Biotechnology and Bioengineering 107 (3), pp. 566 - 581 (2010)
Janke, R.; Genzel, Y.; Freund, S.; Wolff, M. W.; Grammel, H.; Rühmkorf, C.; Seidemann, J.; Wahl, A.; Reichl, U.: Expression, purification, and characterization of a His6-tagged glycerokinase from Pichia farinosa for enzymatic cycling assays in mammalian cells. Journal of Biotechnology 150 (3), pp. 396 - 403 (2010)
Janke, R.; Genzel, Y.; Händel, N.; Wahl, A.; Reichl, U.: Metabolic adaptation of MDCK cells to different growth conditions: Effects on catalytic activities of central metabolic enzymes. Biotechnology and Bioengineering 108 (11), pp. 2691 - 2704 (2011)
Janke, R.; Genzel, Y.; Wetzel, M.; Reichl, U.: Effect of influenza virus infection on key metabolic enzyme activities in MDCK cells. In BMC Proceedings, 5 (S8 ), P129. 22nd European Society for Animal Cell Technology (ESACT) - Meeting on Cell Based Technologies , Vienna, Austria, May 15, 2011 - May 18, 2011. (2011)
 
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