Display library technologies play a central role in protein engineering applications, aiding in the development of novel binding reagents with high affinity interactions to a variety of protein and non-biological targets. To improve the ability to isolate novel binding peptides, robust bacterial display systems have been developed and screened for binding to a range of target proteins.

The first display system relied upon the insertional fusion of a random 15-mer peptide sequence within an extracellular loop of outer membrane protein OmpA of E. coli. A large library of 5×10¹⁰ unique clones was constructed and screened for binding to five unrelated proteins targets: human serum albumin, anti-T7•tag antibody, human C-reactive protein, HIV-1 gp120, and streptavidin. Requiring only two to four rounds of sorting, high-affinity protein binding peptides were isolated for each of the protein targets. A strong amino acid consensus within the selected peptides was apparent for all the target proteins. The OmpA display system provides an efficient process for identifying peptide affinity reagents, but due to the constraints caused by the insertional fusion within the display scaffold, the affinity of the identified peptides decreased when transferred to a soluble format.

To alleviate the imposed constraints of an insertional fusion within OmpA, a bacterial display methodology was developed allowing both N- and C-terminal display and proved to enable rapid screening for protein binding peptides from terminally displayed libraries. To accomplish this, a new scaffold was developed through circular permutation of outer membrane protein OmpX (CPX) to allow terminally displayed peptides to extend from the extracellular surface. CPX display was directly compared to insertional fusion display and enabled isolation of peptides with greater modularity, diversity, and affinity for the target. To further improve the CPX display system, directed evolution was used to create a new scaffold (eCPX) which expedites the display of peptides and allows for the presentation of larger polypeptides than the parental CPX, thus improving the sorting process.

The newly developed display systems provide a highly effective method for screening peptide libraries to rapidly generate ligands with high affinity and specificity to protein targets for an array of biotechnological applications.