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Identifying Conformation-Selective Heavy-Chain-Only Antibodies Against Membrane Proteins by a Thermal-Shift Scintillation Proximity Assay.
- Source :
-
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2020; Vol. 2127, pp. 185-190. - Publication Year :
- 2020
-
Abstract
- 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.
- Subjects :
- Animals
Antibody Specificity
Epitopes chemistry
Epitopes immunology
Epitopes metabolism
Glycine metabolism
Glycine Plasma Membrane Transport Proteins chemistry
Glycine Plasma Membrane Transport Proteins immunology
Glycine Plasma Membrane Transport Proteins metabolism
Humans
Immunoglobulin Heavy Chains chemistry
Immunoglobulin Heavy Chains metabolism
Kinetics
Membrane Proteins metabolism
Protein Binding
Protein Conformation
Substrate Specificity
Temperature
Thermodynamics
Biological Assay methods
Immunoglobulin Heavy Chains isolation & purification
Membrane Proteins chemistry
Membrane Proteins immunology
Subjects
Details
- Language :
- English
- ISSN :
- 1940-6029
- Volume :
- 2127
- Database :
- MEDLINE
- Journal :
- Methods in molecular biology (Clifton, N.J.)
- Publication Type :
- Academic Journal
- Accession number :
- 32112323
- Full Text :
- https://doi.org/10.1007/978-1-0716-0373-4_13