Epitope Mapping by Expression of Restriction Enzyme or PCR Fragments in Bacterial Plasmids

Abstract

Antibodies induced by native antigens often crossreact with denatured antigen or with peptides that contain a segment of the antigen polypeptide chain. The epitopes recognized by these antibodies are linear or sequential, i.e., specified by the linear sequence of the amino acid residues rather than by the protein conformation. Presumably, linear epitopes correspond to flexible parts of the surface of the antigen (1). These epitopes can be localized by synthesis of peptides and testing their antigenicity, i.e., the binding by antibodies. However, this is not practical for antigens of several hundreds of residues. In this chapter, we describe a convenient alternative, the prokaryotic expression of fragments of the coding sequence. In most systems, these fragments are expressed as part of a fusion protein, i.e., coupled to a bacterial polypeptide, which serves as a convenient carrier or provides a tag for affinity purification.