Abstract
BackgroundA 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.MethodsA 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.ResultsTwo linear epitopes, HA peptide 112 – 126 and 162–173, recognized by mAb 12CA5 and pAb 07431, respectively, were identified as the cognate epitopes.ConclusionThis 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.
Highlights
A rapid phage display method for the elucidation of cognate peptide specific ligand for receptors is described
Conventional approaches to elucidate peptide protein interactions include the screening of synthetic sequentially overlapping peptides [1,2], peptide fragments created by enzymatic digestion [3] or expressed gene fragments created by recombinant DNA technologies [4]
The conditions were adjusted in order to create predominantly 50 – 100 bp fragments that were subsequently cloned into the modified pORFES vector [12]
Summary
A rapid phage display method for the elucidation of cognate peptide specific ligand for receptors is described. Conventional approaches to elucidate peptide protein interactions include the screening of synthetic sequentially overlapping peptides [1,2], peptide fragments created by enzymatic digestion [3] or expressed gene fragments created by recombinant DNA technologies [4]. For more than a decade, phage display technology has been applied to elucidate protein-protein interactions. Random peptide libraries have been useful to predict epitope sequence mimics of unknown ligands. The epitopes for antibodies have been predicted from consensus sequences derived from aligning motifs with the original protein sequence of interest [5,6]. When the original ligand is not known, it is possible to predict potential binding consensus sequences. A semi-empirical approach involves displaying randomly generated fragments of the target genes on fd phage [8]. The method has been used to elucidate epitopes for mAbs directed against the bluetongue virus outer capsid protein [9], human plasminogen-activator inhibitor 1 [10], large subunit of Drosophila RNA polymerase II, human p53, and the human cytokeratin 19 [11]
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