Development of a sensitive peptide-based immunoassay: Application to detection of the Jun and Fos oncoproteins
Development of a sensitive peptide-based immunoassay: Application to detection of the Jun and Fos oncoproteins
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DOI：
10.1021/bi952817o
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发表时间：
1996-07-16
期刊：
影响因子：
2.9
通讯作者：
EasterbrookSmith, SB
中科院分区：
文献类型：
--
作者：
Heuer, KH;Mackay, JP;EasterbrookSmith, SB
C-jun和c-Fos属于bZIP类转录激活蛋白，其中许多与细胞的肿瘤转化有关。我们感兴趣的是设计显性负亮氨酸拉链(LZ)多肽，作为一种在体内隔离这些蛋白质的手段，以抑制它们的转录调节活性。为此，我们开发了一种新的免疫分析方法来测量二聚体Jun和Fos络合物的二聚化亲和力。这种基于多肽的酶联免疫吸附试验依赖于这样一个事实，即Jun和Fos优先通过它们的亮氨酸拉链结构域形成异二聚体。用生物素标记重组Jun亮氨酸拉链多肽(天然的JunLZ或V36-->；E点突变体)，并通过亮氨酸拉链相互作用将其与吸附在ELISA盘孔上的FosLZ-谷胱甘肽S-转移酶融合蛋白特异性结合。Jun：随后使用最近开发的基于链霉亲和素的放大系统检测FOS复合体，称为酶复合体放大[Wilson，M.R.，&Easterbrook-Smith，S.B.(1993)anal]。生物化学。209、183-187]。该系统可以检测亚纳摩尔浓度的Jun和Fos，从而可以确定络合物形成的解离常数。在37℃时，天然的JunLZ：FosLZ杂二聚体的解离常数为0.99+/-0.30 nm，而JunLZ(V36E)：FosLZ杂二聚体的解离常数为0.90+/-0.13 mM。这些结果表明，该方法简便、灵敏，可用于快速筛选显性阴性的亮氨酸拉链多肽。
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 mu M. 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.