Paratope-based protein identification by antibody and peptide phage display

In this paper we report a novel application of single-chain antibody fragments (scFv) for protein identification utilizing the inherent information of the paratope for primary structure analysis. Combining the potential of antibody phage display and peptide phage display, selected scFvs are employed to select phage-displayed peptides mimicking an epitope of the protein of interest. Proof of principle is demonstrated by identification of the neuroblastoma protein NB-p260. This protein is recognized by apoptosis-inducing IgM antibodies present in the sera of healthy individuals. Identification of NB-p260 has been hindered by its high molecular weight in the range of 260-280kDa and its instability in purified protein preparations. Employing our approach, we subjected a human synthetic scFv library to selection using sodium dodecyl sulfate-denatured NB-p260. Specific scFvs were further used for selection of a heptapeptide phage display library. From analyzed clones, peptide sequences were identified, two of which could not be related to known proteins by conservative amino acid replacement and one of which, obtained from several clones, could be related to the actin-binding protein ABP278 after two conservative amino acid replacements. The identity of NB-p260 with ABP278 was verified by specific antibodies directed against the N and C termini of ABP278.