Isolation of Antigen-Specific Nanobodies From Synthetic Libraries Using a Protein Selection Strategy That Combines MACS-Based Screening of YSD and FLI-TRAP
Issued Date
2026-01-20
Resource Type
eISSN
23318325
Scopus ID
2-s2.0-105030356645
Journal Title
Bio Protocol
Volume
16
Issue
2
Rights Holder(s)
SCOPUS
Bibliographic Citation
Bio Protocol Vol.16 No.2 (2026)
Suggested Citation
Thaiprayoon A., Chantarasorn Y., Oonanant W., Kasorn A., Longsompurana P., Tapaneeyakorn S., Riangrungroj P., Loison F., Kruse A.C., DeLisa M.P., Waraho-Zhmayev D. Isolation of Antigen-Specific Nanobodies From Synthetic Libraries Using a Protein Selection Strategy That Combines MACS-Based Screening of YSD and FLI-TRAP. Bio Protocol Vol.16 No.2 (2026). doi:10.21769/BioProtoc.5570 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/115425
Title
Isolation of Antigen-Specific Nanobodies From Synthetic Libraries Using a Protein Selection Strategy That Combines MACS-Based Screening of YSD and FLI-TRAP
Author's Affiliation
Harvard Medical School
Cornell University College of Engineering
King Mongkut's University of Technology Thonburi
Faculty of Science, Mahidol University
Thailand National Center for Genetic Engineering and Biotechnology
Thailand National Nanotechnology Center
Vajira Hospital
Cornell Institute of Biotechnology
Cornell University College of Engineering
King Mongkut's University of Technology Thonburi
Faculty of Science, Mahidol University
Thailand National Center for Genetic Engineering and Biotechnology
Thailand National Nanotechnology Center
Vajira Hospital
Cornell Institute of Biotechnology
Corresponding Author(s)
Other Contributor(s)
Abstract
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 Escherichia coli 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.