The yeast two-hybrid system is extremely useful for studying protein:protein interactions. See Chien et al., 1991; Fields and Sternglanz, 1994; Harper et al., 1993; Vojtek et al., 1993; Luban et al., 1993; Li and Fields, 1993; Zang et al, 1993; Golemis and Brent, 1992; Sato et al., 1994; Coghlan et al., 1995; Kalpana et al., 1994; Helps et al., 1994; Yeung et al., 1994; Durfee et al., 1993; Paetkau et al., 1994; Spaargaren and Bischoff, 1994; Ye and Baltimore, 1994. (Full citations of references cited in this and subsequent sections may be found in the Information Disclosure, above.) Variations of the system are available for screening yeast phagemid (Harper et al., 1993; Elledge et al., 1991) or plasmid (Bartel et al., 1993a,b; Finley and Brent, 1994) cDNA libraries to clone interacting proteins, as well as for studying known protein pairs.
The success of the two-hybrid system relies upon the fact that the DNA binding and polymerase activation domains of many transcription factors, such as GAL4, can be separated and then rejoined to restore functionality (Morin et al., 1993).
Yeast strains with integrated copies of various reporter gene cassettes, such as GAL.fwdarw.LacZ, GAL.fwdarw.HIS3 or GAL.fwdarw.URA3 (Bartel et al., 1993a; Harper et al., 1993; Fields and Sternglantz, 1994) are co-transformed with two plasmids, each expressing a different fusion protein. One plasmid encodes a fusion between protein "X" and the DNA binding domain of, for example, the GAL4 yeast transcription activator (Brent and Ptashne, 1985; Ma and Ptashne, 1987; Keegan et al., 1986), while the other plasmid encodes a fusion between protein "Y" and the RNA polymerase activation domain of GAL4 (Keegan et al., 1986). The plasmids are transformed into a strain of the yeast that contains a reporter gene, such as lacZ, whose regulatory region contains GAL4 binding sites. If proteins X and Y interact, they reconstitute a functional GAL4 transcription activator protein by bringing the two GAL4 components into sufficient proximity to activate transcription.
Either hybrid protein alone must be unable to activate transcription of the reporter gene, the DNA-binding domain hybrid, because it does not provide an activation function, and the activation domain hybrid, because it cannot localize to the GAL4 binding sites. Interaction of the two test proteins reconstitutes the function of GAL4 and results in expression of the reporter gene.
The reporter gene cassettes consist of minimal promoters that contain the GAL4 DNA recognition site (Johnson and Davis, 1984; Lorch and Kornberg, 1984) cloned 5' to their TATA box. Transcription activation is scored by measuring either the expression of .beta.-galactosidase or the growth of the transformants on minimal medium lacking the specific nutrient that permits auxotrophic selection for the transcription product, e.g., URA3 (uracil section) or HIS3 (histidine selection). See, Bartel et al., 1993a; Durfee et al., 1993; Fields and Sternglantz, 1994, and U.S. Pat. No. 5,283,173. These and all references cited in this application are hereby incorporated by reference.
The two-hybrid system offers a number of advantages for investigating protein interactions over older methods such as co-immunoprecipitation, crosslinking, and copurification through gradients or chromatographic columns. The biochemical methods have the major disadvantage that interacting proteins are generally known only as bands of a particular relative mobility on a polyacrylamide gel and to progress from these bands to cloned genes is often a difficult undertaking. The assay is performed in vivo, under conditions similar to those in which protein interactions normally occur. Purified target protein or antibody against this protein is not required to detect interactions. And the two-hybrid system appears to be more sensitive than co-immunoprecipitation.
There are no presently available methods for high volume screening for specific inhibitors of protein:protein interactions. This invention describes how to incorporate the two-hybrid system into an effective high volume screen for specific inhibitors of protein:protein interactions.
The documents cited in this section and all the sections below are incorporated by reference unless otherwise indicated.