Journal: Science Advances

Article Title: Multiobjective V H H discovery through integrated high-throughput screening and AlphaFold3-guided structural prioritization

doi: 10.1126/sciadv.aef5325

Figure Lengend Snippet: ( A ) Overview of LNTxs used for the screenings, including a cLNTx (structure predicted with AF3), αCbtx [PDB ID: 1YI5 ], and αBgtx [PDB ID: 1HC9 ] with RMSD between structures annotated. ( B ) Yeast display screenings were performed using biotinylated cLNTx with streptavidin-PE to detect antigen binding and SpyCatcher-GFP to detect V H H surface display. Yeast cells were sorted with FACS into cLNTx binder and nonbinder populations on the basis of these two signals. ( C ) Poly- and monospecific selections were performed using biotinylated αBgtx detected with streptavidin-PE and αCbtx directly labeled with AF488. On the basis of these two signals, three populations were FACS sorted: αBgtx monospecific V H Hs, αCbtx monospecific V H Hs, and αBgtx/αCbtx polyspecific V H Hs. ( D ) Each yeast display selection was deep sequenced, and sequences were clustered by hierarchical clustering using pairwise distances across CDR1-CDR3. Clusters were visualized using t -distributed stochastic neighbor embedding ( t -SNE) plots, enabling selection of relevant variants for each sample. Coloring of populations is the same as in (B) and (C).

Article Snippet: All measurements were performed in Hepes buffer (pH 7.2), and sensors were preincubated in Hepes buffer for 30 min. Biotinylated αBgtx and αCbtx (200 nM final) were immobilized on streptavidin biosensors (Sartorius) to a loading response of ∼1.0 nm.

Techniques: Yeast, Binding Assay, Labeling, Selection