Development of SpyTag-SpyCatcher based sensor surface enabling small molecule detection with biolayer interferometry
Hannan, Md Abdul (2025)
2025
Bioteknologian ja biolääketieteen tekniikan maisteriohjelma - Master's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
Hyväksymispäivämäärä
2025-12-16
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-2025121611757
https://urn.fi/URN:NBN:fi:tuni-2025121611757
Tiivistelmä
The ability to detect how small molecules bind to biological targets is crucial for drug discovery. However, sensitive high-throughput detection remains challenging, especially for low molecular weight inhibitors that produce weak signals. This study aimed to develop and optimize a biolayer interferometry (BLI) platform using the SpyTag-SpyCatcher technology to enhance ligand immobilization, signal stability, and sensitivity for small-molecule interaction analysis. SpyCatcher was genetically fused with target proteins, and biotinylated SpyTag was im-mobilized to the sensor surface using the SwitchAvidin protein, providing a regenerable layer.
SpyCatcher-ERK2 and SpyCatcher-CASK were initially cloned and expressed, but Spy-Catcher-ERK2 was selected as the suitable candidate based on its solubility, purity, and monodispersity. Optimization of bBSA immobilization pH and concentration, SwitchAvidin density, SpyTag polymer expression, and SpyTag valency established a reproducible sensor platform. Penta-SpyTag yielded higher and more stable loading signals compared to Mono-SpyTag, reflecting the benefits of multivalent ligand display.
Staurosporine was used as inhibitor, and the platform produced measurable kinetic parameters consistent with moderate-affinity small-molecule interactions against SpyCatcher-ERK2 in a concentration-dependent manner. Although higher inhibitor concentrations showed non-specific binding in control sensors, the actual binding was distinguishable from the non-specific binding and can be mitigated through further buffer optimization and blocking strategies.
Overall, this study developed a BLI platform with higher sensitivity and regenerability features for detecting small molecule interactions using SpyTag–SpyCatcher technology. The platform serves potential as a versatile tool for fragment screening and early-stage drug discovery and provides a promising solid foundation for future improvements in assay specificity and throughput.
SpyCatcher-ERK2 and SpyCatcher-CASK were initially cloned and expressed, but Spy-Catcher-ERK2 was selected as the suitable candidate based on its solubility, purity, and monodispersity. Optimization of bBSA immobilization pH and concentration, SwitchAvidin density, SpyTag polymer expression, and SpyTag valency established a reproducible sensor platform. Penta-SpyTag yielded higher and more stable loading signals compared to Mono-SpyTag, reflecting the benefits of multivalent ligand display.
Staurosporine was used as inhibitor, and the platform produced measurable kinetic parameters consistent with moderate-affinity small-molecule interactions against SpyCatcher-ERK2 in a concentration-dependent manner. Although higher inhibitor concentrations showed non-specific binding in control sensors, the actual binding was distinguishable from the non-specific binding and can be mitigated through further buffer optimization and blocking strategies.
Overall, this study developed a BLI platform with higher sensitivity and regenerability features for detecting small molecule interactions using SpyTag–SpyCatcher technology. The platform serves potential as a versatile tool for fragment screening and early-stage drug discovery and provides a promising solid foundation for future improvements in assay specificity and throughput.