Integration of phage and yeast display platforms: A reliable and cost effective approach for binning of peptides as displayed on-phage

Abstract

Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine variants on yeast. Flow cytometry was used to detect binding of peptides on-phage to the target on yeast. Once hits were identified, they were synthesized to confirm their binding region(s) by HDX (Hydrogen deuterium exchange) and crystallography. Moreover, we have successfully shown that this approach can be implemented as part of a panning process to deplete non-functional peptides. This technique can be applied to any target that can be successfully displayed on yeast; it narrows down the number of peptides requiring synthesis; and its utilization during selection results in enrichment of peptide population against defined binding regions on the target.

Probing the binding of interleukin-23 to individual receptor components and the IL-23 heteromeric receptor complex in living cells using NanoBRET

“…The results of these competition experiments confirmed that the IL-23:IL12Rβ1 complex had a high affinity for the untagged IL-23 cytokine, K D = 27.2 ± 12.0 pM, which was similar to that measured for IL23-TMR. In the context of a growing list of reported IL23R antagonists ( Cheng et al., 2019 ; Kong et al., 2020 ; Kuchař et al., 2014 ; Pandya et al., 2020 ; Quiniou et al., 2014 ), this assay should be a useful tool for future screening efforts to identify and develop IL-23 receptor inhibitors.…”

Modulating Protein–Protein Interactions by Cyclic and Macrocyclic Peptides. Prominent Strategies and Examples

“…Yeast surface display, alone or in combination with phage display, starts to be an approach for the quick identification of protein binders, with only a few examples to express cyclic peptide derivatives nowadays. Thus, genetically encoded libraries generated by using yeast surface display serve to identify cyclic peptides that bind to lysozyme, interleukin-17 (IL-17) and IL-23 [ 59 , 60 ], and help to envisage this technique as a future strategy toward peptide drug leads and inhibitors of relevant PPIs. Another attractive, completely in vitro method for library production is the mRNA display, which allows the insertion of non-natural amino acid residues and peptide cyclization [ 61 , 62 ], though the application to find PPI binders need to be explored yet.…”

Non-small molecule PROTACs (NSM-PROTACs): Protein degradation kaleidoscope

“…At present, many targets that cannot be targeted by small molecules are degraded by peptide PROTACs ( Table 2 ). Another way to develop targeting peptides with high affinity for POI is to use phage display and yeast display technologies 49 , 50 ,which can find hundreds of target-specific peptides 51 , 52 . More importantly, these technologies do not rely on protein crystal complexes, and can obtain d -configuration peptide targeting warheads, which can avoid the easily degradable characteristics of peptide ligands in the body 53 .…”