Jeffrey J. Rice

Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands

A bacterial display methodology was developed for N‐ and C‐terminal display and demonstrated to enable rapid screening of very large peptide libraries with high precision and efficiency. To overcome limitations of insertional fusion display libraries, a new scaffold was developed through circular permutation of the Escherichia coli outer membrane protein OmpX that presents both N and C termini on the external cell surface. Circularly permuted OmpX (CPX) display was directly compared to insertional fusion display by screening comparable peptide libraries in each format using magnetic and fluorescence activated cell sorting. CPX display enabled in situ measurement of dissociation rate constants with improved accuracy and, consequently, improved affinity discrimination during screening and ranking of isolated clones. Using streptavidin as a model target, bacterial display yielded the well‐characterized HPQ/M motif obtained previously using several alternative peptide display systems, as well as three additional motifs (LI/V CQNVCY, CGWMYF/YxEC, ERCWYVMHWPCNA). Using CPX display, a very high affinity streptavidin‐binding peptide was isolated having a dissociation rate constant koff = 0.002sec−1 even after grafting to the C terminus of an unrelated protein. Comparison of individual clones obtained from insertional fusion and terminal fusion libraries suggests that the N‐terminal display yields sequences with greater diversity, affinity, and modularity. CPX bacterial display thus provides a highly effective method for screening peptide libraries to rapidly generate ligands with high affinity and specificity.

Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides.

Bacterial cell-surface display systems coupled with quantitative screening methods offer the potential to expand protein engineering capabilities. To more fully exploit this potential, a unique bacterial surface display scaffold was engineered to display peptides more efficiently from the surface exposed C- and N-termini of a circularly permuted outer membrane protein. Using directed evolution, efficient membrane localization of a circularly permuted OmpX (CPX) display scaffold was rescued, thereby improving the presentation of diverse passenger peptides on the cell surface. Random and targeted mutagenesis directed towards linkers joining the native N- and C-termini of OmpX coupled with screening by FACS yielded an enhanced CPX (eCPX) variant which localized to the outer membrane as efficiently as the non-permuted parent. Interestingly, enhancing substitutions coincided with a C-terminal motif conserved in outer membrane proteins. Surface localization of various passenger peptides and mini-proteins was expedited using eCPX relative to that achieved with the parent scaffold. The new variant also permitted simultaneous display and labeling of distinct peptides on structurally adjacent C- and N-termini, thus enabling display level normalization during library screening and the display of bidentate or dimeric peptides. Consequently, the evolved scaffold, eCPX, expands the range of applications for bacterial display. Finally, this approach provides a route to improve the performance of cell-surface display vectors for protein engineering and design.

Protease-resistant peptide ligands from a knottin scaffold library.

Peptides within the knottin family have been shown to possess inherent stability, making them attractive scaffolds for the development of therapeutic and diagnostic agents. Given its remarkable stability to proteases, the cyclic peptide kalata B1 was employed as a scaffold to create a large knottin library displayed on the surface of E. coli. A library exceeding 10(9) variants was constructed by randomizing seven amino acids within a loop of the kalata B1 scaffold and screened using fluorescence-activated cell sorting to identify peptide ligands specific for the active site of human thrombin. Refolded thrombin binders exhibited high nanomolar affinities in solution and slow dissociation rates and were able to inhibit thrombins enzymatic activity. Importantly, 80% of a knottin-based thrombin inhibitor remained intact after a 2 h incubation both with trypsin and with chymotrypsin, demonstrating that modifying the kalata B1 sequence did not compromise its stability properties. In addition, the knottin variant mediated 20-fold enhanced affinity for thrombin, when compared to the same seven residue binding epitope constrained by a single disulfide bond. Our results indicate that peptide libraries derived from the kalata B1 scaffold can yield high-affinity protein ligands that retain the remarkable protease resistance associated with the parent scaffold. More generally, this strategy may prove useful in the development of stable peptide ligands suitable for in vivo applications.

Flow Cytometric Sorting of Bacterial Surface‐Displayed Libraries

The protocols herein detail methods for isolating binding peptides from a combinatorial library displayed on the surface of bacterial cells. These methods are appropriate for a variety of display scaffolds and a large range of library sizes, up to ∼5 × 109 or more. Instructions have been provided for isolating peptides that bind to both proteins and non‐protein targets, such as whole cells or inorganic particles. Qualitative analysis by flow cytometry can be exploited for bacterial libraries to characterize a displayed peptides binding properties with a target of interest, and sorting conditions can be tuned to maximize binding affinity. Curr. Protocol. Cytom. 42:4.6.1‐4.6.27.