The Matchmaker 3 Two-Hybrid system from Clontech, a Takara Bio Company, offers an improved approach to two-hybrid screening that reduces false positives and facilitates downstream analyses. Two-hybrid screening is a method of detecting protein-protein interactions that relies on transcriptional activation and reporter genes in yeast. Traditional methods use reporter genes that can be activated in the absence of an interacting protein, leading to false positives. By including an adenine nutritional selection marker in addition to two other reporter genes, researchers can perform a high-stringency screen that virtually eliminates false positives. The kit includes cloning vectors for the bait and library, positive and negative control plasmids, sequencing primers, two yeast strains, carrier DNA, and dropout mix to make selective media.
I used the Matchmaker kit in conjunction with a Matchmaker fetal brain library as part of an effort to characterize a novel protein. Although researchers have the option to construct a library in the vector provided, Clontech sells a number of libraries that are compatible with the system. I co-transformed my bait construct and the library plasmid and identified potential interacting proteins. I initially performed high-stringency screens but isolated very few candidate interacting proteins. I then performed a less stringent screen to detect weaker interactions, which I retested at a higher stringency.
Initially, I found that my transformation efficiency was too low in test screens to proceed with the library scale screen. Ultimately, I purchased competent yeast cells from Clontech (both the AH109 and Y187 strains are available) and was able to move forward. Although purchasing competent yeast added to the expense of the project, it allowed me to perform the screen with only a few micrograms of library plasmid DNA rather than several hundred micrograms. The competent yeast also simplified the procedure, since making competent cells requires several hours of waiting for the yeast to grow. I would recommend that anyone who has problems with transformation efficiency that they cannot resolve with standard troubleshooting consider buying competent AH109 cells. On the other hand, homemade competent cells allow for more tests to confirm the specificity of the interactions, since they are not in limited supply.
There are several advantages to the Matchmaker 3 system. The vectors include epitope tags so that specific antibodies to each potential interactor are not necessary for follow-up studies. The vectors are also compatible with a set of tagged mammalian vectors to facilitate subcloning for confirmation in mammalian cells. In addition, the bait and prey vectors have different antibiotic resistance properties so that standard competent E. coli are suitable to isolate the prey plasmid alone. Furthermore, researchers can verify interactions by yeast mating with the two strains provided. Another bonus is the detailed manual and yeast protocols handbook that are included.
One disadvantage is that the procedure is very costly if one includes the yeast plasmid isolation kits, PCR reagents, yeast media, sequencing expenses, etc. However, researchers can reduce some of these costs by carefully selecting products and not simply purchasing those recommended by the manufacturer. Also, the pre-made libraries need to be amplified in order to obtain enough plasmid DNA. The amplification process is tedious and requires scraping colonies from as many as 500 large agar plates. Also, researchers who choose to buy the competent cells should be aware that the expiration date might be only several weeks after they arrive. The process is also time-consuming, since it involves subcloning steps as well as careful validation of the results. Researchers who opt to construct their own libraries will require additional time. Another potential pitfall to consider is that some bait proteins may not be appropriate for a GAL4 DNA-binding domain-based system. This flaw is a problem with the technique, not with this kit; however, it is still a potential issue that will not be apparent until after an investment of several hundred dollars. Likewise, there is no guarantee that a given screen will identify any interesting interactions.
To summarize, the Matchmaker Two-Hybrid 3 kit is an excellent choice for researchers who are looking for a kit to perform a two-hybrid screen. However, even though it is a kit, the complete process of identifying and validating protein interactions is both time-consuming and expensive. Despite the reduced false positives, confirmation of interactions by coimmunoprecipitation or another method is still essential. Since each protein and DNA construct has its own unique features, there are a number of potential problems that could occur in any given experiment.
Tara Lauriat
Graduate student
Mount Sinai School of Medicine