Cognate peptide-receptor ligand mapping by directed phage display

Abstract

Background: A rapid phage display method for the elucidation of cognate peptide specific ligand for receptors is described. The approach may be readily integrated into the interface of genomic and proteomic studies to identify biologically relevant ligands.

Methods: A gene fragment library from influenza coat protein haemagglutinin (HA) gene was constructed by treating HA cDNA with DNAse I to create 50-100 bp fragments. These fragments were cloned into plasmid pORFES IV and in-frame inserts were selected. These in-frame fragment inserts were subsequently cloned into a filamentous phage display vector JC-M13-88 for surface display as fusions to a synthetic copy of gene VIII. Two well characterized antibodies, mAb 12CA5 and pAb 07431, directed against distinct known regions of HA were used to pan the library.

Results: Two linear epitopes, HA peptide 112-126 and 162-173, recognized by mAb 12CA5 and pAb 07431, respectively, were identified as the cognate epitopes.

Conclusion: This approach is a useful alternative to conventional methods such as screening of overlapping synthetic peptide libraries or gene fragment expression libraries when searching for precise peptide protein interactions, and may be applied to functional proteomics.

Figure 1

Insert modification of pORFES to pORFES IV to permit cloningof blunt-ended fragments. The original pORFES [12, 14] vector was designed to accept inserts as NheI-HindIII fragments. To create pORFES IV, the NheI – HindIII stuffer was replaced with aninsert to include a NaeI site, an additional bp and a (Gly)₄ spacer. The ligation of inserts restoring the correct frame of the β-lactamase confers carbenicillin resistance.

Figure 2

Panning of the HA fragment phage library with mAb 12CA5. The HA fragment phage display library was selected against the anti-haemagglutinin mAb 12CA5 or wells coated with BSA (no antibody). The eluted phage was quantified after each round of panning by plaque assay.

Figure 3

Panning of the HA fragment phage library with mAb 12CA5. The enrichment factor was calculated as the ratio of eluted phage from wells coatedwith antibody or BSA only, respectively, for each round of panning

Figure 4

Panning of the HA fragment phage library with mAb 12CA5. An aliquots of eluted phage after each round of panning were analyzed by filter lifts for binding to mAb 12CA5. The filters were blocked with BSA, incubated with mAb 12CA5 and subsequently detected with goat anti-mouse kappa Ab coupled to alkaline phosphatase.

Figure 5

Panning of the HA fragment phage library with pAb 07431. The HA fragment phage display library was selected against the anti-haemagglutinin pAb 07431 or wells coated with BSA only (no antibody). The Eluted phage was quantified after each round of panning by plaque assay.

Figure 6

Panning of the HA fragment phage library with pAb 07431. The enrichment factor was calculated as the ratio of eluted phage from wells coated with pAb 07431 or BSA only, respectively.

Figure 7

Panning of the HA fragment phage library with pAb 07431. An aliquots of eluted phage after each round of panning were analyzed by filter lifts for binding to pAb 07431. The filters were blocked with BSA, incubated with pAb 07431 and subsequently detected with goat anti-rabbit IgG Ab coupled to alkaline phosphatase.