Development of a sensitive peptide-based immunoassay: application to detection 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 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 degrees C was determined to be 0.99 +/- 0.30 nM, while that for JunLZ(V36E):FosLZ heterodimer was 0.90 +/- 0.13 microM. 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.
Heuer
K H
KH
Department of Biochemistry, University of Sydney, Australia.
Mackay
J P
JP
Podzebenko
P
P
Bains
N P
NP
Weiss
A S
AS
King
G F
GF
Easterbrook-Smith
S B
SB
D000595:Amino Acid Sequence; D001710:Biotin; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D016759:Oncogene Protein p65(gag-jun) / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D016761:Oncogene Proteins v-fos / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000276:immunology* / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity
D016759:Oncogene Protein p65(gag-jun); D016761:Oncogene Proteins v-fos; D010455:Peptides; D011993:Recombinant Fusion Proteins; D001710:Biotin; D005982:Glutathione Transferase