Epitope mapping by negative selection of randomized antigen libraries displayed on filamentous phage.
Since most antibodies directed against protein antigens recognize epitopes composed of several discontinuous segments of the polypeptide chain, attempts to delineate the amino acids constituting these epitopes with the use of linear peptides have generally been unsuccessful. Here, a method is described based on error-prone PCR, phage display and negative selection, whereby amino acid residues constituting the functional epitope are identified in the context of the native protein. First a library of randomized antigen variants containing most single, double and triple amino acid mutants generated by single nucleotide substitutions is produced by error-prone PCR amplification of the DNA sequence encoding the protein antigen. The phage-displayed library is then negatively selected for epitope loss mutants by passing through an affinity matrix derivatized with a specific antibody and positively selected for retention of function. This method was applied to the mapping of the epitopes of two murine monoclonal antibodies (MA-7H11 and MA-3G10) on staphylokinase, a 136 amino acid plasminogen activator secreted by some strains of Staphylococcus aureus. After two negative/positive selection cycles, DNA sequencing of several clones revealed preferential amino acid mutations at positions 35 and 130 (with MA-7H11), and at positions 62, 66 and 136 (with MA-3G10). Affinity measurements of staphylokinase variants carrying single amino acid mutations at these positions confirmed their contribution to the free energy of binding to MA-7H11 and MA-3G10. This approach may be useful for isolating mutants with altered antigenic or functional properties and in general to map critical regions for protein-protein interactions.
Jespers
L
L
Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, KULeuven, Leuven, Belgium.
Jenné
S
S
Lasters
I
I
Collen
D
D
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology*; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000940:Antigenic Variation; D000942:Antigens, Bacterial / Q000276:immunology*; D001483:Base Sequence; D018604:Epitope Mapping / Q000379:methods*; D005343:Fibrinolytic Agents / Q000276:immunology*; D017911:Inoviridae / Q000235:genetics; D008666:Metalloendopeptidases / Q000276:immunology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019151:Peptide Library; D012641:Selection, Genetic; D017386:Sequence Homology, Amino Acid
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D005343:Fibrinolytic Agents; D019151:Peptide Library; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus