THE 2-HYBRID SYSTEM - A METHOD TO IDENTIFY AND CLONE GENES FOR PROTEINS THAT INTERACT WITH A PROTEIN OF INTEREST
THE 2-HYBRID SYSTEM - A METHOD TO IDENTIFY AND CLONE GENES FOR PROTEINS THAT INTERACT WITH A PROTEIN OF INTEREST
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DOI：
10.1073/pnas.88.21.9578
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发表时间：
1991-11-01
影响因子：
11.1
通讯作者：
FIELDS, S
中科院分区：
文献类型：
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作者：
CHIEN, CT;BARTEL, PL;FIELDS, S
我们描述了一种方法，检测能够与已知蛋白质相互作用的蛋白质，并导致这些相互作用的蛋白质的克隆基因的立即可用性。构建质粒以编码两种杂合蛋白。一个杂交体由融合到已知蛋白质的酵母转录激活蛋白GAL 4的DNA结合结构域组成;另一个杂交体由融合到由酵母基因组DNA片段文库编码的蛋白质序列的GAL 4激活结构域组成。已知蛋白质和由文库质粒之一编码的蛋白质之间的相互作用导致含有GAL 4结合位点的报告基因的转录激活。我们使用这种方法与酵母SIR 4蛋白，这是参与酵母交配型信息的转录抑制。(i)我们使用双杂交系统来证明SIR 4可以形成同源二聚体。(ii)由SIR 4的C末端组成的小结构域被证明足以介导这种相互作用。(iii)我们筛选了一个文库来检测可以与SIR 4 C-末端结构域相互作用的杂合蛋白，并从该文库中鉴定了SIR 4。通过在激活结构域质粒中构建合适的cDNA文库，这种方法可以容易地扩展到哺乳动物蛋白。
We describe a method that detects proteins capable of interacting with a known protein and that results in the immediate availability of the cloned genes for these interacting proteins. Plasmids are constructed to encode two hybrid proteins. One hybrid consists of the DNA-binding domain of the yeast transcriptional activator protein GAL4 fused to the known protein; the other hybrid consists of the GAL4 activation domain fused to protein sequences encoded by a library of yeast genomic DNA fragments. Interaction between the known protein and a protein encoded by one of the library plasmids leads to transcriptional activation of a reporter gene containing a binding site for GAL4. We used this method with the yeast SIR4 protein, which is involved in the transcriptional repression of yeast mating type information. (i) We used the two-hybrid system to demonstrate that SIR4 can form homodimers. (ii) A small domain consisting of the C terminus of SIR4 was shown to be sufficient to mediate this interaction. (iii) We screened a library to detect hybrid proteins that could interact with the SIR4 C-terminal domain and identified SIR4 from this library. This approach could be readily extended to mammalian proteins by the construction of appropriate cDNA libraries in the activation domain plasmid.