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Flow cytometry as a useful tool for process development: rapid evaluation of expression systems.
Anant Patkar1, Natarajan Vijayasankaran, Dan W Urry
1Department of Chemical Engineering and Materials Science, University of Minnesota, 151 Amundson Hall, 421 Washington Avenue S.E., Minneapolis, MN 55455-0312, USA.
Journal of Biotechnology
|January 5, 2002
Summary
Flow cytometry effectively monitors microbial fermentation for process development. This method tracks protein production and plasmid-free cells in recombinant Escherichia coli, optimizing expression systems.
Area of Science:
- Biotechnology
- Microbial Physiology
- Process Development
Background:
- Flow cytometry is a standard technique for single-cell microbial physiology studies.
- Its application in bioprocess development for recombinant protein production is less explored.
Purpose of the Study:
- To demonstrate the utility of flow cytometry in monitoring and optimizing fermentation processes for recombinant protein production.
- To evaluate different expression systems and induction strategies in Escherichia coli.
Main Methods:
- Utilized recombinant Escherichia coli strains expressing a protein-based polymer.
- Employed single-cell right angle light scatter to detect intracellular product formation (inclusion bodies).
- Used translational fusions with green fluorescent protein (GFP) to monitor product accumulation and plasmid-free cell fraction (PFCF).
Main Results:
- Single cell light scatter correlated with intracellular product formation, indicating inclusion body presence.
- GFP fusions successfully monitored product accumulation and PFCF.
- Evaluated T7, T7-lac, and P(BAD) promoter-based expression systems, with P(BAD) showing superior performance.
Conclusions:
- Flow cytometry is a valuable tool for bioprocess development, enabling real-time monitoring of fermentation.
- The P(BAD) promoter system demonstrated superior expression efficiency compared to T7-based systems in this context.