Extended yeast surface display linkers enhance the enrichment of ligands in direct mammalian cell selections.

Abstract

Selections of yeast-displayed ligands on mammalian cell monolayers benefit from high target expression and nanomolar affinity, which are not always available. Prior work extending the yeast-protein linker from 40 to 80 amino acids improved yield and enrichment but is hypothesized to be below the optimal length, prompting evaluation of an extended amino acid linker. A 641-residue linker provided enhanced enrichment with a 2-nM affinity fibronectin ligand and 105 epidermal growth factor receptors (EGFR) per cell (14±2 vs. 8±1, P=0.008) and a >600-nM affinity ligand, 106 EGFR per cell system (23±7 vs. 0.8±0.2, P=0.004). Enhanced enrichment was also observed with a 310-nM affinity affibody ligand and 104 CD276 per cell, suggesting a generalizable benefit to other scaffolds and targets. Spatial modeling of the linker suggests that improved extracellular accessibility of ligand enables the observed enrichment under conditions not previously possible.