Identifying Conformation-Selective Heavy-Chain-Only Antibodies Against Membrane Proteins by a Thermal-Shift Scintillation Proximity Assay
| Autor: | Peter Stohler, Roger J. P. Dawson |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
biology Chemistry Drug discovery Transporter Small molecule In vitro Solute carrier family 03 medical and health sciences 030104 developmental biology 0302 clinical medicine Scintillation proximity assay Membrane protein Glycine transporter 1 biology.protein Biophysics 030217 neurology & neurosurgery |
| Zdroj: | Methods in Molecular Biology ISBN: 9781071603727 |
| DOI: | 10.1007/978-1-0716-0373-4_13 |
| Popis: | Over the last decades, the use of heavy-chain-only antibodies has received growing attention in academia and industry as research and diagnostic tools as well as therapeutics. Their generation has improved with the help of innovative new methods such as the sybody technology; however, identifying conformation-selective compounds against membrane proteins remains a major challenge. In this chapter, we apply a thermal shift scintillation proximity assay (SPA-TS) to identify sybodies from an in vitro display campaign with the ability to selectively stabilize the inhibitor-bound conformation of the human solute carrier (SLC) family transporter SC6A9 (GlyT1). Using detergent-purified GlyT1 protein and a tritium-labeled glycine uptake inhibitor small molecule, we find sybody candidates that increase the apparent melting temperature in SPA-TS by several degrees. The thermal shift stabilizes the GlyT1-inhibitor complex and qualifies the sybodies for structural studies and inhibitor-selective small molecule screening assays. The SPA-TS assay in its current form is adaptable to any antibody discovery campaign for membrane proteins and permits the generation of highly valuable tools in most stages of drug discovery and development. |
| Databáze: | OpenAIRE |
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