Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation
Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation
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DOI：
10.1073/pnas.94.16.8405
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发表时间：
1997-08-05
影响因子：
11.1
通讯作者：
Blau, HM
中科院分区：
文献类型：
--
作者：
Rossi, F;Charlton, CA;Blau, HM
我们提出了一种监测完整真核细胞内蛋白质-蛋白质相互作用的方法，这应该增加我们对控制细胞增殖和分化的调节电路以及这些过程在癌症等疾病状态下如何出错的理解，由与互补β-半乳糖苷酶（β-gal）缺失突变体融合的感兴趣蛋白质组成的嵌合蛋白允许对细胞内蛋白质复合物进行新的分析。在这种方法中，由非功能性弱互补β-gal肽（Delta）强制相互作用产生的β-gal活性alpha 和 Delta omega) 作为嵌合体非 β-gal 部分相互作用程度的衡量标准，为了测试 lacZ 顺反子内互补的应用，使用了在雷帕霉素存在下形成复合物的蛋白质，这些蛋白质：FRAP 和 FKBP12 分别与 Delta alpha 和 Delta omega 合成为融合蛋白，酶促 beta-gal 活性用于监测雷帕霉素诱导的嵌合体的形成FRAP/FKBP12 蛋白复合物以时间和剂量依赖性方式进行评估，通过组织化学、生物化学和荧光激活细胞分选测定进行评估。这种方法可能被证明是体外免疫沉淀和交联方法以及体内酵母双杂交和荧光能量转移系统的有价值的辅助手段。它还可以允许在生物学相关背景下直接评估特定蛋白质二聚化相互作用，定位于它们发生的细胞区室中，以及在竞争蛋白质的环境。
We present an approach for monitoring protein-protein interactions within intact eukaryotic cells, which should increase our understanding of the regulatory circuitry that controls the proliferation and differentiation of cells and how these processes go awry in disease states such as cancer, Chimeric proteins composed of proteins of interest fused to complementing beta-galactosidase (beta-gal) deletion mutants permit a novel analysis of protein complexes within cells, In this approach, the beta-gal activity resulting from the forced interaction of nonfunctional weakly complementing beta-gal peptides (Delta alpha and Delta omega) serves as a measure of the extent of interaction of the non-beta-gal portions of the chimeras, To test this application of lacZ intracistronic complementation, proteins that form a complex in the presence of rapamycin were used, These proteins: FRAP and FKBP12, were synthesized as fusion proteins with Delta alpha and Delta omega, respectively, Enzymatic beta-gal activity served to monitor the formation of the rapamycin-induced chimeric FRAP/FKBP12 protein complex in a time- and dose-dependent manner, as assessed by histochemical, biochemical, and fluorescence-activated cell sorting assays, This approach may prove to be a valuable adjunct to in vitro immunoprecipitation and crosslinking methods and in vivo yeast two-hybrid and fluorescence energy transfer systems, It may also allow a direct assessment of specific protein dimerization interactions in a biologically relevant context, localized in the cell compartments in which they occur, and in the milieu of competing proteins.