DEVELOPMENT OF A SENSITIVE PEPTIDE-BASED IMMUNOASSAY - APPLICATION TODETECTION OF THE JUN AND FOS ONCOPROTEINS

c-Jun and c-Fos belong to the bZIP class of transcriptional activator proteins, many of which have been implicated in the neoplastic transfo rmation of cells. We are interested in engineering dominant-negative l eucine 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 pe ptide-based ELISA relies on the fact that Jun and Fos preferentially f orm heterodimers via their leucine zipper domains. Recombinant Jun leu cine zipper peptides (either native JunLZ or a V36-->E point mutant) w ere labeled with biotin and specifically bound through a leucine zippe r interaction to a FosLZ-glutathione S-transferase fusion protein adso rbed onto the wells of an ELISA tray. Jun:Fos complexes were subsequen tly detected using a recently developed streptavidin-based amplificati on system known as enzyme complex amplification [Wilson, M. R., & East erbrook-Smith, S. B. (1993) Anal. Biochem. 209, 183-187]. This ELISA s ystem can detect subnanomolar concentrations of Jun and Fos, thus allo wing determination of the dissociation constants for complex formation . The dissociation constant for formation of the native JunLZ:FosLZ he terodimer at 37 degrees C was determined to be 0.99 +/- 0.30 nM, while that for JunLZ(V36E):FosLZ heterodimer was 0.90 +/- 0.13 mu M. These results demonstrate that the novel peptide-based ELISA described herei n is simple and sensitive and can be used to rapidly screen for potent ial dominant-negative leucine zipper peptides.