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        Epitope Mapping of Protein Antigens by Expression-PCR (E-PCR)

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        It is often the case that one has a cloned gene for a protein and a monoclonal antibody (MAb) that recognizes an epitope within that protein. Determination of the amino acid sequences constituting an epitope recognized by the MAb can be problematic. Producing ever and ever smaller fragments of the protein in vivo using expression systems, such as bacteria, yeast, or baculovirus, leads to problems of having the expression system influence the protein, i.e., correct folding, glycosylation, and degradation. In some cases, the foreign protein may in fact be toxic to the cell, thereby making synthesis impossible. In addition, each new fragment in the study has to be constructed, usually by PCR from the gene, cloned, and then sequenced to check for fidelity against the parent sequence to avoid PCR errors. To detect the newly expressed protein by immunoprecipitation, radiolabeled amino acids are usually incorporated into the new product, but at the same time this label goes into all other host proteins. This can be further complicated if MAb crossreactivity or coprecipitation occurs. Most of these problems can be avoided by epitope mapping using proteins synthesized by expression-PCR (E-PCR) (1 ). E-PCR is a rapid and simple method for the in vitro production of proteins without having to go through the rigors of cloning. The resulting radiochemically pure proteins are useful for a variety of purposes that include studies on the subunit structure of proteins, epitope mapping, and protein mutagenesis.
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