(119c) Engineering High-Affinity Antibodies Using Inner Membrane Display of Translocation Intermediates

Karlsson, A. J., Cornell University
Lim, H., Cornell University
Rocco, M. A., Cornell University
DeLisa, M. P., Cornell University

The bacterial twin-arginine translocation (Tat) pathway is capable of exporting native-like proteins to the periplasm. Here, we discovered that overexpressed Tat substrates form two distinct, long-lived translocation intermediates that are readily detected by immunolabeling methods. Formation of the early translocation intermediate Ti-1, which exposes the N- and C-terminus to the cytoplasm, was observed in the absence of a functional Tat signal peptide or intact Tat translocase and did not require the substrate to be correctly folded. The later translocation intermediate Ti-2, which exhibits a bitopic topology with the N-terminus in the cytoplasm and C-terminus in the periplasm, required both a functional signal peptide and translocase and was only detected when the substrate was correctly folded. These results support a model whereby Tat substrates initially insert unassisted into the lipid bilayer and interactions with the Tat translocase occur only in later stages of the translocation process. We also observed that Ti-2 formation resulted in the display of correctly folded substrates on the periplasmic face of the Escherichia coli inner membrane. Based on this observation, we developed a technology for membrane-anchored expression of translocation intermediates that enabled isolation of enhanced ligand-binding proteins from combinatorial libraries displayed on the inner membrane. Following two rounds of mutagenesis and selection, we were able to dramatically improve the intracellular folding and antigen binding affinity of a human single-chain antibody. This approach has several advantages for library screening, including the unique involvement of the Tat folding quality control mechanism that ensures only native-like proteins are displayed, thus eliminating poorly folded sequences from the screening process.