Engineering of site-directed antisera against vertebrate calmodulin by using synthetic peptide immunogens containing an immunoreactive site

Site-directed antisera against vertebrate calmodulin were elicited in rabbits by injection of a synthetic immunogen containing the pentadecapeptide Gly-Gln-val-Asn-Tyr-Glu-Glu-Phe-Val-Gln-Met-Met-Thr-Ala-Lys-OH, which corresponds to residues 134-148 of vertebrate calmodulin. A major immunoreactive region (residues 127-144) of calmodulin is found in the COOH-terminal structural domain and an immunoreactive site for one antiserum is contained in the heptapeptide Asn-Tyr-Glu-Glu-Phe-Val-Gln-NH₂, which corresponds to residues 137-143 of vertebrate calmodulin. This immunoreactive heptapeptide was conjugated to a carrier protein by adding a cysteine residue to the NH₂ terminus of the peptide and coupling the Cys-heptapeptide to the carrier through the thiol group of the cysteine residue. Injection of this Cys-heptapeptide-protein conjugate into rabbits yielded antisera that react with the heptapeptide but not with native calmodulin. Thus, the immunoreactive heptapeptide that is exposed on the surface of calmodulin is immunogenic, but it is not sufficient to elicit antibodies that react with native calmodulin. However, when the Cys-pentadecapeptide corresponding to residues 134-148 and containing the immunoreactive heptapeptide sequence was conjugated to a carrier protein and injected into rabbits, antisera were elicited that react with the intact calmodulin molecule. The affinities and specificites of these antisera for calmodulin are similar to those of antisera elicited by injection of the intact protein and are sufficient for their use in radioimmunoassays. These results indicate that the successful engineering of site-directed antisera against proteins by using synthetic peptide immunogens may require an appropriate intramolecular environment that allows the peptide region to closely approximate the spatial orientation it adopts in the intact protein.