Protein engineers have been using display technologies for many years to develop novel binding ligands with high affinity interactions to a variety of targets. The most common display methodology for more than a decade has been phage display, which has proven to be a valuable tool for protein design. Bacterial display offers the advantages of being quantitative, rapid analysis of library populations and individual clones, and allowing for immediate feed back for kinetic screening of libraries. We have developed an optimized bacterial display system that allows for simultaneous N- and C- terminal display. To achieve this, the monomeric outer membrane protein OmpX was circularly permuted by genetically fusing the native termini, previously oriented towards the periplasmic space, and opening the protein at the extracellular loop two, causing new termini to be located external to the cell. The initial circularly permuted OmpX (CPX) had a decreased display ability compared to the native OmpX, but after two hours of expression the display of peptides could be easily detected. Using directed evolution CPX was optimized to achieve expression capabilities equivalent to the non-permuted sequence, and allow for rapid display of large peptides and mini-proteins. Substitutions that contributed to the increased performance were located within the linker sequence introduced to join the native N- and C- termini and within the native C-terminal region, a motif widely conserved in transmembrane proteins. Using this optimized display scaffold, various peptides and mini-proteins were tested for efficient display and found to have improved expression characteristics when compared to the original CPX. Using terminally displayed peptide libraries, novel peptide ligands were identified for various protein targets. The terminal peptide display scaffold also allows for efficient screening and optimization of protein ligands.

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