Development of a Sensitive Peptide-Based Immunoassay: Application to Detection of the Jun and Fos Oncoproteins

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Abstract
C-Jun and c-Fos belong to the bZIP class of transcriptional activator proteins, many of which have been implicated in the neoplastic transformation of cells. We are interested in engineering dominant-negative leucine zipper (LZ) peptides as a means of sequestering these proteins in vivo in order to suppress their transcriptional regulatory activity. Toward this end, we have developed a novel immunoassay for measuring the dimerization affinities of dimeric Jun and Fos complexes. This peptide-based ELISA relies on the fact that Jun and Fos preferentially form heterodimers via their leucine zipper domains. Recombinant Jun leucine zipper peptides (either native JunLZ or a V36→E point mutant) were labeled with biotin and specifically bound through a leucine zipper interaction to a FosLZ−glutathione S-transferase fusion protein adsorbed onto the wells of an ELISA tray. Jun:Fos complexes were subsequently detected using a recently developed streptavidin-based amplification system known as enzyme complex amplification [Wilson, M. R., & Easterbrook-Smith, S. B. (1993) Anal. Biochem. 209, 183−187]. This ELISA system can detect subnanomolar concentrations of Jun and Fos, thus allowing determination of the dissociation constants for complex formation. The dissociation constant for formation of the native JunLZ:FosLZ heterodimer at 37 °C was determined to be 0.99 ± 0.30 nM, while that for JunLZ(V36E):FosLZ heterodimer was 0.90 ± 0.13 μM. These results demonstrate that the novel peptide-based ELISA described herein is simple and sensitive and can be used to rapidly screen for potential dominant-negative leucine zipper peptides.