In general, the genes for two fusion proteins, one containing the DNA-binding domain with protein X of interest and the other having the activation domain with protein Y of interest, are co-transfected into an appropriate yeast strain. If proteins X and Y physically interact with each other, this interaction brings DB and AD of the transcription factor together and results in the transcription of a reporter gene.
An advantage of this system is that it requires only the cDNA, full length or partial, of the genes of interest. This system can also be used to screen libraries for potential partners of a known protein. The two-hybrid system amplifies even weak interactions and is thus very sensitive.
The best argument in favor of this system is the speed with which several signal transduction pathways have been elucidated by using this method. The yeast two-hybrid system can also be utilized to identify inhibitors of protein-protein interactions.
Some disadvantages of the yeast two-hybrid system include the requirement for post-translational modifications for some interactions that might not happen in yeast, the presence of mammalian proteins that might be toxic in yeast, inadequate representation while screening libraries, and the appearance of false positives.
The Prolexys HyNet system makes use of plasmid libraries encoding human proteins fused to either of the two domains to create bait and prey hybrid proteins. Bait proteins are fused to the BD, and this hybrid binds directly to the promoter of the reporter genes.
In contrast, the prey proteins are fused to the AD. These hybrids are recruited to the promoter only if the human protein portions of the bait-and-prey proteins bind each other. Libraries of bait-and-prey proteins are created in haploid yeast of opposite mating types. Mating between the haploids results in diploids, which contain both bait-and-prey protein as well as the reporter genes.
If bait-and-prey interact, a complex is formed that localizes the AD to the promoter of the reporter gene resulting in expression of the reporter. Yeast colonies only grow if the reporter gene is expressed, so the resulting colonies all represent fruitful protein-protein interactions between bait and prey.
The Prolexys HySpec process is a directed process of protein expression, purification, and high throughput analysis of protein-protein interactions based on pull down experiments and mass spectrometry. Multiprotein complexes (MPCs) are formed in vitro by incubating purified tagged bait proteins with mammalian cell extracts under conditions that allow protein interactions to take place.
After a series of washes to remove nonspecific contaminants, tandem affinity purification (TAP) is used to isolate the MPCs. In addition, MPCs can also be isolated directly from human cells expressing the tagged protein bait, again by using TAP. The individual proteins of the MPCs are identified by making use of the power of mass spectrometry.
This technique can be modified to identify the interacting partners of drugs and drug candidates by using these small molecule agents as the bait. Identification of the drug can lead to an understanding of the molcular mechanisms of action and the design of protein complexes (targets) associated with newer and better drugs, remarks Dr. Boniface.
Another high throughput method to identify interacting proteins is to use protein arrays. ProteinOne (www.proteinone. com) has developed a protein array for identifying interacting partners to a known protein, says Neil Sharma, senior scientist.
The array contains 3040 immobilized proteins and can be used to screen for binding to the protein of interest. The ProteinOne array offers proteins that are highly pure and functionally active. One advantage of this system is that several variants of the protein of interest can be generated and immobilized on the array and be used as bait to study protein-protein interactions and the effects of the mutations on the interactions.
The limitations of the system include the availability of only about 200 mammalian proteins in a purified and functionally active state. The detection system used is radioactive and other substitutes are being currently researched.