Quartz-Crystal Microbalance Biosensor-Based Biopanning to Study Protein-Drug Interactions
筆記録
To study protein-drug interactions, begin with a quartz-crystal microbalance, QCM, sensor chip comprising piezoelectric ceramic material coated with a thin gold layer acting as an electrode.
Overlay the electrode with test drug solution, forming a uniform layer.
Insert the sensor chip into the QCM apparatus. Immerse it into a buffer-containing cuvette. Apply an electric field, causing ceramic crystals to oscillate at a fixed frequency based on the drug molecules' mass over the sensor chip.
Add a T7 phage library containing a mixture of genetically engineered phages displaying peptide variants fused with the phages' capsid protein. The phages displaying a specific peptide bind to drug molecules with high affinity and get immobilized on the sensor chip. This panning of T7 phages increases the mass on the sensor chip, resulting in crystal oscillation decrease over time.
Post-equilibration, transfer the phage-bound sensor chip into a humid petri dish. Overlay the chip with a bacterial solution and incubate. Phages infect the bacteria and replicate, producing specific peptide-expressing phages that get released in the solution.
Recover these phages and analyze the drug-affinity-selected peptides' amino acid sequence. Compare this peptide's amino acid sequence with the target protein.
The region with high similarity represents the potential drug interaction site crucial for the drug-protein interaction.
