An Amber-Encoding Helper Phage for More Efficient Phage Display of Noncanonical Amino Acids

In the past two decades, phage display has emerged as a powerful technique for the identification of antibodies and peptide ligands for therapeutic targets. Using the amber suppression-based noncanonical amino acid (ncAA) mutagenesis approach, we and others have shown that the chemical space in phage display can be significantly expanded for drug discovery. However, the use of amber codon in phages results in poor phage yields and requires tedious processes to enrich amber codon-containing (amber obligate) phage clones. In this work, we demonstrate the development of a novel helper phage, CMa13ile40, for rapid and continuous enrichment of amber obligate phage clones and efficient production of ncAA-containing phages. CMa13ile40 was constructed by the insertion of a Candidatus Methanomethylophilus alvus pyrrolysyl-tRNA synthetase/PylT gene cassette into a helper phage genome. The afforded novel helper phage allowed for a continuous amber codon enrichment strategy for two different phage display libraries and demonstrated a 100-fold increase in selectivity for packaging of library plasmids in comparison with original helper phage plasmids. To demonstrate the applicability of the system, CMa13ile40 was used to create two phage-displayed peptide libraries containing two separate ncAAs, Nε -tert-butoxycarbonyl-lysine (BocK) and Nε -allyloxycarbonyl-lysine (AllocK), respectively. These were then used to identify peptide ligands that bind to the extracellular domain of ZNRF3, a membrane-bound E3 ligase. Each selection showed differential enrichment of unique sequences dependent upon the ncAA used. Using biolayer interferometry, enriched peptides from both selections were confirmed to have low micromolar affinity for ZNRF3 and this affinity is dependent on the presence of the ncAA used for selection. Our results clearly show that ncAAs in phages provide unique interactions for selection of peptides that are different from each other and from canonical amino acids. As an effective tool for phage display, we believe that CMa13ile40 can be broadly applied to a wide variety of applications.