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.
Show moreYear 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