Dmitrova M, Younès-Cauet G, Oertel-Buchheit P, Porte D, Schnarr M, Granger-Schnarr M
Institut de Biologie Moleculaire et Cellulaire, UPR 9002 du CNRS, Strasbourg, France.
Mol Gen Genet. 1998 Jan;257(2):205-12. doi: 10.1007/s004380050640.
Interactions between proteins affect a wide variety of biological processes, such as signal transduction and control of gene expression. In order to facilitate the study of protein-protein interactions we have developed a new method for specifically detecting the heterodimerization of two heterologous proteins in the bacterium Escherichia coli. The assay is based on the simultaneous use of protein fusions with an altered specificity and a wild-type LexA repressor DNA-binding domain. We have tested this system with two well known eukaryotic dimerization domains (the Fos and Jun leucine zippers). The two interacting proteins were, respectively, fused to a wild-type and a mutant LexA DNA-binding domain. Their hetero-association is specifically measured by the transcriptional repression of a reporter gene (lacZ) controlled by a hybrid operator containing a wild-type half-site (CTGT) and a mutated operator half-site (CCGT). The hybrid operator/lacZ construct was integrated into the chromosome of the reporter strain (SU202) to avoid possible artefacts due to variations in plasmid copy number. This method should be particularly useful in those cases where one or both partners are also able to form homodimers, since the assay described here is sensitive only to the formation of heterodimers. Furthermore, this assay gives rise to a screenable red/white phenotype on MacConkey-lactose indicator plates, allowing for a genetic study of the specificity of the interaction.
蛋白质之间的相互作用影响着各种各样的生物过程，如信号转导和基因表达的调控。为了便于研究蛋白质-蛋白质相互作用，我们开发了一种新方法，用于特异性检测大肠杆菌中两种异源蛋白质的异源二聚化。该检测方法基于同时使用具有改变特异性的蛋白质融合体和野生型LexA阻遏物DNA结合结构域。我们用两个著名的真核二聚化结构域（Fos和Jun亮氨酸拉链）对该系统进行了测试。两种相互作用的蛋白质分别与野生型和突变型LexA DNA结合结构域融合。它们的异源缔合通过由包含野生型半位点（CTGT）和突变型操纵子半位点（CCGT）的杂交操纵子控制的报告基因（lacZ）的转录抑制来特异性测定。杂交操纵子/lacZ构建体整合到报告菌株（SU202）的染色体中，以避免由于质粒拷贝数变化而可能产生的假象。这种方法在其中一个或两个伙伴也能够形成同源二聚体的情况下应该特别有用，因为这里描述的检测方法仅对异源二聚体的形成敏感。此外，该检测在麦康凯乳糖指示平板上产生可筛选的红/白表型，从而可以对相互作用的特异性进行遗传学研究。
