ArticlebioRxiv : the preprint server for biology2025
Selection of nanobodies against liponanoparticle-embedded membrane proteins by yeast surface display.
Article in bioRxiv : the preprint server for biology, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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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
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