Nested sets of protein fragments and their use in epitope mapping: characterization of the epitope for the S4D5 monoclonal antibody binding to receptor associated protein

Abstract

The present report describes a new general procedure by which linear and some structure-dependent epitopes may be mapped in a protein antigen using a nested set of protein fragments prepared from partial proteolysis products of a recombinant protein. Briefly, the antigen, fused to an affinity tag, is partially fragmented and affinity sorted under denaturing conditions to produce a nested set of polypeptides, consisting of N- (or C-)terminal fragments. Immunoblots of SDS-PAGE fractionated sets of fragments are therefore directly readable in terms of molecular mass — i.e., approximate sequence positions — that identify sequence segments harbouring an epitope and any additional structural elements, required to maintain epitope conformation. Blots of N- and C-terminal nested sets of polypeptide fragments representing the human receptor associated protein (RAP) were prepared and probed with mAb S4D5 (Moestrup and Gliemann, 1991). Fragments 1–177 and 94–323 were the shortest fragments detected by the antibody, suggesting the presence of an epitope within the 94–177 segment. Independent mapping based on recombinant fragments of the RAP homologue, rat Heymann nephritis antigen, confirmed that the epitope resides in the Pro 115-Asp 177 segment. The model study demonstrates the utility of nested sets of protein fragments as fast and inexpensive tools for epitope mapping.