Chien C T, Bartel P L, Sternglanz R, Fields S
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook 11794.
Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9578-82. doi: 10.1073/pnas.88.21.9578.
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.
我们描述了一种方法，该方法可检测能够与已知蛋白质相互作用的蛋白质，并能立即获得这些相互作用蛋白质的克隆基因。构建质粒以编码两种杂交蛋白。一种杂交蛋白由酵母转录激活蛋白GAL4的DNA结合结构域与已知蛋白融合而成；另一种杂交蛋白由GAL4激活结构域与酵母基因组DNA片段文库编码的蛋白质序列融合而成。已知蛋白与文库质粒之一编码的蛋白之间的相互作用导致含有GAL4结合位点的报告基因的转录激活。我们将此方法应用于酵母SIR4蛋白，该蛋白参与酵母交配型信息的转录抑制。（i）我们使用双杂交系统证明SIR4可以形成同二聚体。（ii）由SIR4的C末端组成的一个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以检测能够与SIR4 C末端结构域相互作用的杂交蛋白，并从该文库中鉴定出SIR4。通过在激活结构域质粒中构建合适的cDNA文库，这种方法可以很容易地扩展到哺乳动物蛋白。
