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Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects
Published on: February 18, 2014
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A method for measuring binding constants using unpurified in vivo biotinylated ligands
Anastassia K Pogoutse1, Christine Chieh-Lin Lai1, Nicholas Ostan2
1Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Analytical Biochemistry
|February 23, 2016
Summary
This study demonstrates that purified proteins are not required for accurate biomolecular interaction analysis. Using in vivo biotinylation with BirA ligase in soluble lysates allows for high-quality binding data via bio-layer interferometry (BLI).
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- Accurate kinetic and binding constants for biomolecular interactions typically necessitate pure protein preparations and labeled ligands.
- Despite advancements in high-throughput technologies like bio-layer interferometry (BLI) for measuring protein-protein interactions, protein purification remains a significant bottleneck.
- Existing methods for analyzing biomolecular interactions are often limited by the laborious and time-consuming process of protein purification.
Purpose of the Study:
- To investigate the feasibility of obtaining high-quality binding data from unpurified protein samples.
- To evaluate the utility of in vivo biotinylation using BirA ligase for simplifying biomolecular interaction analysis.
- To determine if endogenous BirA ligase activity in Escherichia coli is sufficient for producing biotinylated proteins for binding experiments.
Main Methods:
- Utilizing soluble lysate fractions containing in vivo biotinylated proteins.
- Employing bio-layer interferometry (BLI) sensors for simultaneous measurement of multiple biomolecular interactions.
- Assessing binding data quality without further protein purification steps after in vivo biotinylation.
Main Results:
- High-quality binding data can be obtained from soluble lysate fractions without protein purification.
- In vivo biotinylation of proteins using BirA ligase enables direct application to BLI sensors.
- Endogenous BirA ligase activity in Escherichia coli is sufficient for biotinylating proteins, eliminating the need for co-overexpression.
Conclusions:
- In vivo biotinylation simplifies the process of preparing samples for biomolecular interaction analysis.
- This method bypasses the need for extensive protein purification, saving time and resources.
- The findings suggest a more efficient approach to studying protein-protein interactions using readily available cellular lysates.
