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Updated: Apr 23, 2026
Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments
Published on: October 15, 2016
Optimizing recombinant antibodies for intracellular function using hitchhiker-mediated survival selection.
Dujduan Waraho-Zhmayev1, Bunyarit Meksiriporn2, Alyse D Portnoff2
1School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA Biological Engineering Program, Faculty of Engineering, King Mongkut's University of Technology Thonburi, 126 Pracha-utid Road, Bangmod, Toongkru, Bangkok 10140, Thailand.
The FLI-TRAP method uses bacterial growth to rapidly isolate improved intracellular antibodies (intrabodies). This technique enhances antibody stability and antigen-binding affinity without needing antigen purification.
Area of Science:
- Molecular Biology
- Biotechnology
- Protein Engineering
Background:
- The twin-arginine translocation pathway's 'hitchhiker' mechanism enables detection of protein interactions in living bacterial cells.
- Previous adaptations allowed for genetic selection of protein interactions within the cytoplasm of Escherichia coli.
Purpose of the Study:
- To extend the FLI-TRAP method for rapid isolation of intracellular antibodies (intrabodies) with enhanced properties.
- To demonstrate the utility of FLI-TRAP for improving intrabody stability and antigen-binding affinity.
Main Methods:
- Utilized the FLI-TRAP (Fattest Ligation-Independent Trapping and Rapid Amplification of Plasmid) system, adapted from the bacterial twin-arginine translocation pathway.
- Employed a survival-based enrichment strategy requiring bacterial growth on high antibiotic concentrations to select for functional intrabodies.
- Applied FLI-TRAP to isolate improved intrabodies against the yeast Gcn4p transcription factor and the alpha-synuclein non-amyloid component.
Main Results:
- A single round of FLI-TRAP enrichment yielded intrabody variants with >10-fold increased intracellular stability and yield against Gcn4p.
- An intrabody targeting the alpha-synuclein non-amyloid component exhibited an ~8-fold improvement in antigen-binding affinity.
- The method successfully isolated intrabodies with superior traits without antigen purification or immobilization.
Conclusions:
- The FLI-TRAP method is a powerful tool for intracellular antibody engineering, enabling rapid selection for enhanced stability and affinity.
- This approach offers a significant advancement for developing therapeutic and research intrabodies directly within a cellular context.
- FLI-TRAP bypasses traditional antibody purification steps, streamlining the development process for intracellular antibodies.
