Selection of nanobodies against liponanoparticle‐embedded membrane proteins by yeast‐surface display
Year: 2025
Type: article
Abstract: Single-domain antibodies, known as nanobodies (Nbs), are widely used in structural biology, therapeutics, and as molecular probes in biology and biotechnology. Nbs towards soluble proteins are routinely developed via alpaca immunization or directed evolution in yeast cell-surface display. However, for membrane proteins, the targets are generally detergent-solubilized, and there remains a need for Nb development methods against membrane proteins in a native-like membrane environment. To address this need, we present a protocol for Nb selection via extraction of membrane proteins into amphiphilic polymers such as styrene-maleic acid to produce purified membrane proteins in stable liponanoparticles. Proof of generality is demonstrated by applying the pipeline to four membrane-resident enzymes of differing fold, oligomerization state, and membrane topology (reentrant membrane helix, transmembrane, membrane-associated). Following screening for optimal stabilization into liponanoparticles, Nbs were selected against four target proteins from glycoconjugate biosynthesis pathways. The selected Nbs showed high affinity and selectivity towards their target proteins with K D apparent values ranging from 15 nM to 200 nM, depending on the Nb-protein conjugate. In accordance with their tight binding, various Nb-protein complexes were found to be stable to size-exclusion chromatography purification. The Nbs were also amenable to sortase-mediated ligation, enabling their conversion into molecular probes for the target membrane protein. The ability to select for such high-affinity Nb against membrane proteins in SMALP will facilitate their widespread application in cell biology and biomedical applications.
Source: Protein Science
DOI: 10.1002/pro.70293
Institutions: Massachusetts Institute of Technology, Boston University
Language: English
Subfield: Radiology, Nuclear Medicine and Imaging
Field: Medicine
Domain: Health Sciences
Open Access status: green
Awards: G5886465041, G5985125515, G9000139889, G9000193359, Deciphering the Principles of Membrane-Associated Glycan Assembly for Glycoconjugate Biosynthesis, Structure and function of the monotopic phosphoglycosyl transferase superfamily: Initiators of biosynthesis of complex bacterial glycoconjugates
OpenAlex ID: W4414162072