KARYOPLASMIC INTERACTION SELECTION STRATEGY - A GENERAL STRATEGY TO DETECT PROTEIN PROTEIN INTERACTIONS IN MAMMALIAN-CELLS
KARYOPLASMIC INTERACTION SELECTION STRATEGY - A GENERAL STRATEGY TO DETECT PROTEIN PROTEIN INTERACTIONS IN MAMMALIAN-CELLS
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DOI：
10.1073/pnas.89.17.7958
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发表时间：
1992-09-01
影响因子：
11.1
通讯作者：
DANG, CV
中科院分区：
文献类型：
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作者：
FEARON, ER;FINKEL, T;DANG, CV
我们描述了一种用于研究哺乳动物细胞中蛋白质-蛋白质相互作用的策略和试剂，称为核质相互作用选择策略（KISS）。利用这种策略，通过酵母转录激活因子GAL 4的功能活性的重建和GAL 4调节的报告基因的所得转录来鉴定特定的蛋白质-蛋白质相互作用。GAL 4功能的重建是由两种嵌合蛋白之间的特异性相互作用引起的：一种含有GAL 4的DNA结合结构域;另一种含有转录激活结构域。如果两种嵌合蛋白可以形成复合物，重建GAL 4的DNA结合和转录激活功能，则报告基因的转录发生。使用KISS系统，我们证明了特定的相互作用的序列从三个不同的蛋白质对，复杂的细胞质。 此外，我们证明，报告基因编码的细胞表面或耐药标志物可以被特异性激活的蛋白质-蛋白质相互作用的结果。有了这些选择标记，KISS系统可用于筛选专门的cDNA库以鉴定新的蛋白质相互作用。
We describe a strategy and reagents for study of protein-protein interactions in mammalian cells, termed the karyoplasmic interaction selection strategy (KISS). With this strategy, specific protein-protein interactions are identified by reconstitution of the functional activity of the yeast transcriptional activator GAL4 and the resultant transcription of a GAL4-regulated reporter gene. Reconstitution of GAL4 function results from specific interaction between two chimeric proteins: one contains the DNA-binding domain of GAL4; the other contains a transcriptional activation domain. Transcription of the reporter gene occurs if the two chimeric proteins can form a complex that reconstitutes the DNA-binding and transcriptional activation functions of GAL4. Using the KISS system, we demonstrate specific interactions for sequences from three different pairs of proteins that complex in the cytoplasm. In addition, we demonstrate that reporter genes encoding cell surface or drug-resistance markers can be specifically activated as a result of protein-protein interactions. With these selectable markers, the KISS system can be used to screen specialized cDNA libraries to identify novel protein interactions.