A White Plaque, Associated with Genomic Deletion, Derived from M13KE-Based Peptide Library Is Enriched in a Target-Unrelated Manner during Phage Display Biopanning Due to Propagation Advantage

Abstract

The nonspecific enrichment of target-unrelated peptides during biopanning remains a major drawback for phage display technology. The commercial Ph.D.^TM-7 phage display library is used extensively for peptide discovery. This library is based on the M13KE vector, which carries the lacZα sequence, leading to the formation of blue plaques on IPTG-X-gal agar plates. In the current study, we report the isolation of a fast-propagating white clone (displaying WSLGYTG peptide) identified through screening against a recombinant protein. Sanger sequencing demonstrated that white plaques are not contamination from environmental M13-like phages, but derive from the library itself. Whole genome sequencing revealed that the white color of the plaques results from a large 827-nucleotide genomic deletion. The phenotypic characterization of propagation capacity through plaque count- and NGS-based competitive propagation assay supported the higher propagation rate of Ph-WSLGYTG clone compared with the library. According to our data, white plaques are likely to arise endogenously in Ph.D. libraries due to mutations in the M13KE genome and should not always be viewed as exogenous contamination. Our findings also led to the conclusion that the deletion observed here might be an ancestral mutation already present in the naïve library, which causes target-unrelated nonspecific enrichment of white clone during biopanning due to propagation advantage.

Keywords: M13KE; Ph.D.TM-7 peptide library; biopanning; competitive propagation; genomic deletion; lacZα sequence; phage display; propagation-related TUP; white plaque.

Figure 1

Plaques from WSLGYTG-displaying phage (left plate) and Ph.D.^TM-7 peptide library (right plate) plated on separate plates and a close-up of a white and a blue plaque. WSLGYTG-displaying clone produces white plaques, whereas library clones produce blue plaques.

Figure 2

DNA sequence alignment between a fraction of the genomes of WT-M13 (at the top), WT-M13KE (in the middle) and eight isolated white plaques (at the bottom). All eight clones show evidence of arising from the library as they contain nucleotides (marked in blue) that differentiate them from the corresponding nucleotides in WT-M13 (marked in green). Additionally, all the eight white clones express the peptide WSLGYTG and the linker GGGS (marked in red), which are not present in the genomes of both WT-M13 and WT-M13KE. The nucleotide numbers refer to WT-M13KE genome. Compared to WT-M13, the nucleotide numbers are lower by 1 in WT-M13KE due to the deletion of T at position 1565. The alignment analysis was performed in SnapGene software (from Insightful Science; available at snapgene.com).

Figure 3

Evaluation of WSLGYTG-displaying white clone binding to the target protein mCD4. Target-coated wells (0, 5 µg/well) were incubated with phage clones (White-WSLGYTG, WT-M13KE, positive control, and no phage). The plate was incubated with anti-M13 antibody conjugated with HRP and color development was achieved by adding TMB and quenching with H₂SO₄. The absolute absorbance values (450 nm) were reference subtracted (540 nm) and binding ratio was determined through dividing clone binding by CD4-coated/PBS-coated wells (OD450 (CD4)/OD450 (PBS)). The white-WSLGYTG clone does not show significant binding to CD4 once compared with positive control. PBS: Phosphate buffered saline (no target), WT-M13KE: Ph.D.^TM-7 library clone without displayed peptide, ns: non-significant.

Figure 4

Schematic representation M13KE genome indicating large intergenic deletion (red) revealed by whole genome sequencing. The deletion was detected in positions 5879–6705 (827 nucleotides), causing deletion of the segment encoding lacZα gene. The M13KE genomic information was downloaded from New England Biolabs repository [12] and the figure was prepared using SnapGene and BioRender.

Figure 5

NGS-based competitive propagation assay. Equal titers of white-WSLGYTG clone and Ph.D.^TM-7 library were inoculated in early log E.coli culture and amplified for 4.5 h. At three time points, samples were withdrawn and the percentage of WSLGYTG-displaying clone compared to random peptide library clones was calculated based on NGS data. A substantially higher propagation rate was found for WSLGYTG clone compared to the library (representing an average propagation rate of the clones in the library).