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Genetically engineered binding proteins as biosensors for fermentation and cell culture
Xudong Ge1, Leah Tolosa, Jen Simpson
1Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250, USA.
Biotechnology and Bioengineering
|November 5, 2003
Summary
Engineered biosensors using glucose-binding protein (GBP) and glutamine-binding protein (QBP) show high sensitivity for ligand detection in bioprocessing. These stable, fluorophore-labeled proteins offer potential for high-throughput applications with minimal sample volumes.
Area of Science:
- Biotechnology
- Biosensor Development
- Protein Engineering
Background:
- Engineered binding proteins from E. coli, glucose-binding protein (GBP) and glutamine-binding protein (QBP), were modified with single cysteine mutations.
- These mutations allow for the introduction of environmentally sensitive fluorophores (ANS for GBP, acrylodan for QBP) for signal transduction.
Purpose of the Study:
- To investigate the signal-transduction properties and potential applications of engineered GBP and QBP biosensors.
- To evaluate the performance of these biosensors in various bioprocessing scales and compare them with existing analytical methods.
Main Methods:
- Fluorophore labeling of engineered proteins (GBP with ANS, QBP with acrylodan).
- Characterization of protein-ligand binding kinetics and response ranges.
- Testing biosensor performance in yeast and E. coli fermentations, and cell cultures across different scales.
- Comparison of biosensor results with a YSI 2700 Chemistry Analyzer.
Main Results:
- Both GBP and QBP responded to their respective ligands (glucose and glutamine) in the micromolar range.
- The biosensors demonstrated stability, with storage possible at 4°C for over 5 months.
- Encapsulation of proteins led to delayed response and recovery times, which could be mitigated by using smaller dialysis tubing.
- The GBP biosensor successfully monitored glucose in fermentations where the YSI analyzer failed.
- The QBP biosensor was effective in small-scale cell cultures where the YSI's sample volume requirement was prohibitive.
Conclusions:
- Engineered GBP and QBP biosensors offer sensitive, rapid, and reversible detection of their target analytes.
- These biosensors exhibit excellent stability and are suitable for diverse bioprocessing applications, including high-throughput screening.
- Their low sample volume requirement and high sensitivity make them advantageous over conventional methods for microscale applications.