Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS

Sloth, Ane Beth; Bakhshinejad, Babak; Stavnsbjerg, Camilla; Rossing, Maria; Kjær, Andreas

doi:10.3390/ijms24065396

Cited by 2 publications

References 38 publications

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Screening macrocyclic peptide libraries by yeast display allows control of selection process and affinity ranking

Linciano,

Mazzocato,

Romanyuk

et al. 2024

Preprint

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Macrocyclic peptides provide an attractive modality for drug development due to their ability to bind challenging targes, their small size, and amenability to powerfulin vitroevolution techniques such as phage or mRNA display. While these technologies proved capable of generating and screening extremely large libraries and yielded ligands to already many targets, they often do not identify the best binders within a library due to the difficulty of monitoring performance and controlling selection pressure. Furthermore, only a small number of enriched ligands can typically be characterised due to the need of chemical peptide synthesis and purification prior to characterisation. In this work, we address these limitations by developing a yeast display-based strategy for the generation, screening and characterisation of structurally highly diverse disulfide-cyclised peptides. Analysis and sorting by quantitative flow cytometry enabled monitoring the performance of millions of individual macrocyclic peptides during the screening process and allowed us identifying macrocyclic peptide ligands with affinities in the low micromolar to high picomolar range against five highly diverse protein targets. X-ray analysis of a selected ligand in complex with its target revealed optimal shape complementarity, large interaction surface, constrained peptide backbones and multiple inter- and intra-molecular interactions, rationalising the high affinity and exquisite selectivity. The novel technology described here offers a facile, quantitative and cost-effective alternative to rapidly and efficiently generate and characterise fully genetically encoded macrocycle peptide ligands with sufficiently good binding properties to even therapeutically relevant targets.

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Screening macrocyclic peptide libraries by yeast display allows control of selection process and affinity ranking

Linciano,

Mazzocato,

Romanyuk

et al. 2024

Preprint

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

Sell,

Bakhshinejad,

Sinkjaer

et al. 2024

CIMB

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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.

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Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS

Cited by 2 publications

References 38 publications

Screening macrocyclic peptide libraries by yeast display allows control of selection process and affinity ranking

Screening macrocyclic peptide libraries by yeast display allows control of selection process and affinity ranking

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

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