Regenerable Biosensors for Small-Molecule Kinetic Characterization Using SPR

Regenerable Biosensors for Small-Molecule Kinetic Characterization Using SPR

Description

A key activity in small-molecule drug discovery is the characterization of compound–target interactions. Surface plasmon resonance (SPR) is a flexible technique for this purpose, with a wide affinity range (micromoles to picomoles), low protein requirements, and the ability to characterize the kinetics of compound binding. However, a key requirement of SPR is the immobilization of the target protein to the surface of the sensor chip. The most commonly used immobilization techniques (covalent immobilization, streptavidin–biotin) are irreversible in nature, which can afford excellent baseline stability but impose limitations throughput for slowly dissociating compounds or unstable targets. Reversible immobilization (e.g., His-tag–Ni-NTA) is possible but typically precludes accurate quantification of slow dissociation kinetics due to baseline drift.Here we present our investigation of three immobilization strategies (dual-His-tagged target protein, His-tagged streptavidin, and switchavidin) that combine the robustness of irreversible immobilization with the flexibility of reversible immobilization. Each has its own advantages and limitations, and while a universal immobilization procedure remains to be found, these strategies add to the immobilization toolbox that enables previously out-of-scope applications. Such applications are highlighted in two examples that greatly increased throughput for the kinetic characterization of potent kinase inhibitors and kinetic profiling of covalent inhibitors.
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Year of publication

2020

Authors

SAGE Journals - Publisher

Vesa Hytönen - Creator

Unknown organization

Rupam Jha - Contributor

Anders Gunnarsson - Creator

Christopher J. Stubbs - Creator

Eleanor Taylor-Newman - Creator

Geoffrey Holdgate - Creator

Göran Dahl - Creator

Philip B. Rawlins - Creator

Stefan Geschwindner - Creator

Wei-Chao Lee - Creator

Other information

Fields of science

Medical biotechnology

Language

English

Open access

Open

License

Creative Commons Attribution 4.0 International (CC BY 4.0)

Keywords

Medical biotechnology
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