(110c) Enhancing FLI-TRAP for Rapid Isolation of Solubility and Affinity Matured Antibodies

We successfully developed FLI-TRAP (Functional Ligand-binding Identification by Tat-based Recognition of Associating Proteins), a simple and versatile selection method for isolating interacting proteins.  The technique utilizes the unique ability of the Escherichia coli twin-arginine translocation (Tat) pathway to transport non-covalent complexes of two folded polypeptides, termed the ‘hitchhiker’ mechanism.  In the FLI-TRAP assay, one polypeptide consists of a fusion between an N-terminal Tat signal peptide (ssTorA) and the protein to be screened for interaction, and the second of a fusion between a known or putative partner protein and mature TEM-1 β-lactamase (Bla).  In solubility and/or affinity maturation processes, mutations that improve solubility often compromise function and vice versa.  FLI-TRAP, on the other hand, couples selection for solubility and binding activity in a single step.  Through selection of yeast GCN4 binders from a randomized CDR-H3 anti-GCN4 scFv library, we previously validated the power of FLI-TRAP in isolating interacting, soluble, non-aggregating, and protease-resistance proteins pairs.  Here, we show that this technique can be easily extended to screen naïve libraries for isolation of intracellular antibodies against protein antigens of interest. By simply increasing the selection stringency, FLI-TRAP can be further extended as a tool for solubility and/or affinity maturation of selected clones.  Finally, we have developed ‘competitive FLI-TRAP’ whereby selection is performed under competitive binding in vivo and thus favors the isolation of affinity-matured clones. Collectively, these advances reveal the versatility of the FLI-TRAP method and highlight its potential for routine selection of high-affinity binding proteins (e.g., antibody-antigen) without the need for purification or immobilization of the binding target.