Development of a Sensitive Peptide-Based Immunoassay: Application to Detection of the Jun and Fos Oncoproteins
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- 作者：Katja H. Heuer ; Joel P. Mackay ; Philip Podzebenko ; Naresh P. S. Bains ; Anthony S. Weiss ; Glenn F. King ; and Simon B. Easterbrook-Smith
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：July 16, 1996
- 年：1996
- 卷：35
- 期：28
- 页码：9069 - 9075
- 全文大小：264K
- 年卷期：v.35,no.28(July 16, 1996)
- ISSN：1520-4995
文摘
c-Jun and c-Fos belong to the bZIP class oftranscriptional activator proteins, many of whichhave been implicated in the neoplastic transformation of cells. Weare interested in engineering dominant-negative leucine zipper (LZ) peptides as a means of sequestering theseproteins in vivo in order to suppresstheir transcriptional regulatory activity. Toward this end, wehave developed a novel immunoassay formeasuring the dimerization affinities of dimeric Jun and Fos complexes.This peptide-based ELISA relieson the fact that Jun and Fos preferentially form heterodimers via theirleucine zipper domains. RecombinantJun leucine zipper peptides (either native JunLZ or a V36
rarr.gif">E pointmutant) were labeled with biotin andspecifically bound through a leucine zipper interaction to aFosLZ-glutathione S-transferase fusionproteinadsorbed onto the wells of an ELISA tray. Jun:Fos complexes weresubsequently detected using a recentlydeveloped streptavidin-based amplification system known as enzymecomplex amplification [Wilson, M.R., & Easterbrook-Smith, S. B. (1993) Anal.Biochem. 209, 183-187]. This ELISA systemcan detectsubnanomolar concentrations of Jun and Fos, thus allowing determinationof the dissociation constantsfor complex formation. The dissociation constant for formation ofthe native JunLZ:FosLZ heterodimerat 37 
C was determined to be 0.99 ± 0.30 nM, while that forJunLZ(V36E):FosLZ heterodimer was0.90 ± 0.13 
r.gif">M. These results demonstrate that the novelpeptide-based ELISA described herein issimple and sensitive and can be used to rapidly screen for potentialdominant-negative leucine zipperpeptides.
rarr.gif">E pointmutant) were labeled with biotin andspecifically bound through a leucine zipper interaction to aFosLZ-glutathione S-transferase fusionproteinadsorbed onto the wells of an ELISA tray. Jun:Fos complexes weresubsequently detected using a recentlydeveloped streptavidin-based amplification system known as enzymecomplex amplification [Wilson, M.R., & Easterbrook-Smith, S. B. (1993) Anal.Biochem. 209, 183-187]. This ELISA systemcan detectsubnanomolar concentrations of Jun and Fos, thus allowing determinationof the dissociation constantsfor complex formation. The dissociation constant for formation ofthe native JunLZ:FosLZ heterodimerat 37 C was determined to be 0.99 ± 0.30 nM, while that forJunLZ(V36E):FosLZ heterodimer was0.90 ± 0.13 r.gif">M. These results demonstrate that the novelpeptide-based ELISA described herein issimple and sensitive and can be used to rapidly screen for potentialdominant-negative leucine zipperpeptides.