Screening combinatorial peptide libraries in complex mixtures for applications in therapeutic delivery and molecular diagnostics

Display technologies are powerful tools to isolate peptide reagents for applications ranging from protein separations to diagnostics. Screening methods have focused on achieving the desired affinity rather than specificity, which can be even more important since many of the engineered peptides must be functional in complex mixtures such as serum. High specificity is therefore crucial to eliminate undesired interactions especially for therapeutic targeting and diagnostic applications. Phage display is the most widely used display technology, but bacterial display may hold advantages including ease of use and quantitative screening via fluorescence activated cell sorting (FACS). For the work presented here, bacterial display libraries were quantitatively screened in complex mixtures to achieve the desired specificity for the intended applications and to explore its advantages for applications in which phage display has traditionally been used.;An auto-fluorescent bacterial display peptide library was used to isolate red blood cell (RBC) binding ligands which were used to attach nanoparticles to the RBC surface to develop novel long circulating drug delivery vehicles. Bacterial display was also employed to isolate tissue targeting ligands in vivo. Finally, multi-color FACS was used to quantitatively isolate highly-specific peptides and further optimize their specificity for a target antibody present at a 10,000--100,000 fold dilution in serum IgG. The method was employed to screen serum antibodies of patients with Celiac Disease to identify potential biomarker candidates. The method developed here enables quantitative screening and evolution of specificity, enhancing the current repertoire of screening methods available in protein engineering.