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Monolith weak affinity chromatography for μg-protein-ligand interaction study
Lucile Lecas1, Jérôme Randon1, Alain Berthod1
1Université de Lyon, CNRS, Université Claude Bernard Lyon 1, Institut des Sciences Analytiques, UMR 5280, 5 rue de la Doua, F-69100, Villeurbanne, France.
Journal of Pharmaceutical and Biomedical Analysis
|January 18, 2019
Summary
New affinity monolith columns enable rapid protein-ligand interaction studies using minimal sample. The streptavidin-biotin method on organic supports offers superior protein immobilization and activity for accurate affinity measurements.
Area of Science:
- Biochemistry and Analytical Chemistry
- Chromatography and Separation Science
- Biomolecular Interaction Analysis
Background:
- Investigating protein-ligand interactions is crucial for drug discovery and understanding biological processes.
- Traditional methods often require large amounts of purified proteins and can be time-consuming.
- Developing efficient and sensitive methods for affinity chromatography is essential for high-throughput screening.
Purpose of the Study:
- To develop novel affinity monolith columns for efficient protein-ligand affinity investigations.
- To compare different monolithic supports and bio-functionalization strategies for optimal protein immobilization.
- To validate the developed method for accurate determination of binding affinities (Kd values).
Main Methods:
- In-situ synthesis of silica and glycidylmethacrylate based monoliths.
- Grafting of human serum albumin (HSA) using epoxy-amine, Schiff Base, and streptavidin-biotin methods.
- Evaluation using frontal analysis on a capillary electrophoresis instrument with an external pressure device.
Main Results:
- Organic monoliths with the streptavidin-biotin approach yielded the highest active protein content (40 pmol HSA/column) with minimal non-specific interactions (<10%) and high protein activity (84%).
- The immobilization process was rapid (10 min), UV-monitored, and automated, requiring only 3 μg of HSA.
- Validated Kd measurements for three known HSA ligands and demonstrated applicability to the HSP90 protein domain.
Conclusions:
- Affinity monolith columns, particularly organic ones utilizing the streptavidin-biotin strategy, provide a highly efficient platform for protein-ligand interaction studies.
- The method allows for automated, low-pressure chromatography with minimal reagent consumption and high sensitivity.
- This technique is robust, reproducible, and applicable to various proteins of pharmaceutical interest, with stable columns storable for up to 3 months.
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