Using NGS to Uncover the Corruption of a Peptide Phage Display Selection.

Phage display has been widely used to identify peptides binding to a variety of biological targets. In the current work, we planned to select novel peptides targeting CD4 through screening of a commercial phage display library (New England Biolabs Ph.D.^TM-7). After three rounds of biopanning, 57 phage clones were Sanger-sequenced. These clones represented 30 unique peptide sequences, which were subjected to phage ELISA, resulting in the identification of two potential target binders. Following peptide synthesis, downstream characterization was conducted using fluorescence plate-based assay, flow cytometry, SPR, and confocal microscopy. The results revealed that neither of the peptides identified in the Sanger-based phage display selection exhibited specific binding toward CD4. The naïve library and the phage pool recovered from the third round of biopanning were then subjected to next-generation sequencing (NGS). The results of NGS indicated corruption of the selection output by a phage already known as a fast-propagating clone whose target-unrelated enrichment can shed light on the misidentification of target-binding peptides through phage display. This work provides an in-depth insight into some of the challenges encountered in peptide phage display selection. Furthermore, our data highlight that NGS, by exploring a broader sequence space and providing a more precise picture of the composition of biopanning output, can be used to refine the selection protocol and avoid misleading the process of ligand identification. We hope that these findings can describe some of the complexities of phage display selection and offer help to fellow researchers who have faced similar situations.