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.
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 Pro115-Asp177 segment. The model study demonstrates the utility of nested sets of protein fragments as fast and inexpensive tools for epitope mapping.
Ellgaard
L
L
Department of Chemistry, University of Aarhus, Denmark.
Holtet
T L
TL
Moestrup
S K
SK
Etzerodt
M
M
Thøgersen
H C
HC
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis*; D026541:Heymann Nephritis Antigenic Complex; D006801:Humans; D015151:Immunoblotting; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008562:Membrane Glycoproteins / Q000276:immunology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology*; D010449:Peptide Mapping / Q000379:methods*; D051381:Rats; D011971:Receptors, Immunologic / Q000276:immunology*
D000911:Antibodies, Monoclonal; D001324:Autoantigens; D000939:Epitopes; D026541:Heymann Nephritis Antigenic Complex; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011971:Receptors, Immunologic