Isolation of Antigen-Specific Nanobodies From Synthetic Libraries Using a Protein Selection Strategy That Combines MACS-Based Screening of YSD and FLI-TRAP

Although protein-protein interactions (PPIs) are central to nearly all biological processes, identifying and engineering high-affinity intracellular binders remains a significant challenge due to the complexity of the cellular environment and the folding constraints of proteins. Here, we present a two-stage complementary platform that combines magnetic-activated cell sorting (MACS)-based yeast surface display with functional ligand-binding identification by twin-arginine translocation (Tat)-based recognition of associating proteins (FLI-TRAP), a bacterial genetic selection system for efficient screening, validation, and optimization of PPIs. In the first stage, MACS-based yeast display enables the rapid high-throughput identification of candidate binders for a target antigen from a large synthetic-yeast display library through extracellular interaction screening. In the second stage, an antigen-focused library is subcloned into the FLI-TRAP system, which exploits the hitchhiker export process of the <i>Escherichia coli</i> Tat pathway to evaluate binder-antigen binding in the cytoplasm. This stage is achieved by co-expressing a Tat signal peptide-tagged protein of interest with a β-lactamase-tagged antigen target, such that only binder-antigen pairs with sufficient affinity are co-translocated into the periplasm, thus rendering the bacterium β-lactam antibiotic resistant. Because Tat-dependent export requires fully folded and soluble proteins, FLI-TRAP further serves as a stringent in vivo filter for intracellular compatibility, folding, and stability. Therefore, this approach provides a powerful and cost-effective pipeline for discovering and engineering intracellular protein binders with high affinity, specificity, and functional expression in bacterial systems. This workflow holds promise for several applications, including synthetic biology and screening of theragnostic proteins and PPI inhibitors. Key features • Combines a single round of MACS enrichment with FLI-TRAP for high-throughput Nb discovery. • Reduces time and resource demands compared to traditional workflows involving multiple rounds of MACS/FACS. • Enables in vivo selection of high-affinity, functional binders via Tat-dependent export linked to β-lactam resistance, correlating binding affinity and solubility with antibiotic resistance.