Our Matchmaker systems are highly advanced tools for identifying and characterizing novel protein-protein interactions (PPIs). Our latest and most powerful incarnation, the Matchmaker Gold Yeast Two-Hybrid System, adds a sensitive Aureobasidin A (AbA; Takesako et al. 1991) antibiotic resistance marker to two nutritional reporters and blue/white color selection. This results in a four-reporter system with the easiest, most stringent yeast two-hybrid (Y2H) screening strategy available (Figure 1).

Aureobasidin A effectively kills yeast, but when the AUR1-C gene is turned on by a positive interaction between GAL4-hybrid proteins, the yeast become AbA-resistant. Secondary confirmation of positive clones employs four reporters regulated by three different GAL4-responsive promoters. This effectively eliminates false positives and leaves you with greater numbers of genuine positives. Quality screening results like these, as well as our simple Mate & Plate library screening protocol, save you time and make your search for PPIs faster, easier, and more fruitful.

Y2H systems exploit the modular nature of eukaryotic transcription factors, which consist of a sequence-specific DNA-binding domain (DNA-BD) and an RNA Pol II-recruiting transcription activation domain (AD; Fields and Song 1989; Chien et al. 1991). In Matchmaker systems, a known protein of interest is fused to the DNA-BD of the yeast GAL4 transcription factor to create a “bait” protein. Interacting partner proteins, often derived from a library, are expressed as fusions to the AD of yeast GAL4, to create “prey” proteins (Figure 1). When pairs of interacting bait and prey fusion proteins are coexpressed in a yeast cell, GAL4 function is restored and the interacting fusion proteins are able to activate transcription of the reporter genes. In Matchmaker Gold, yeast clones that harbor interacting protein pairs can then be identified by the presence of the four reporters (Figure 2). In library screens, the plasmids containing the coding sequences for the library-derived prey proteins can be rescued from the surviving yeast clones and subjected to further analysis and sequencing.

Aureobasidin A selection eliminates background

Matchmaker Gold systems are unique because they use the AUR1-C gene as a novel reporter that confers resistance to AbA, a potent antibiotic toxic to S. cerevisiae. Resistance to this highly stable antifungal depsipeptide (see technical note) allows straightforward Y2H library screening to be achieved without the optimization required when using nutritional selection alone. HIS3-based selection can produce high numbers of background colonies in primary screens, whereas AbA very effectively kills yeast cells not expressing the AUR1-C reporter. As a result, even low-stringency primary screens using AbA are quite definitive and produce a high percentage of genuinely positive clones, without the interference of background colonies.

Four reporters identify genuine positives

The stringency of Matchmaker Gold lies in the use of four selective reporter genes: AUR1-C, HIS3, ADE2, and MEL1 (α-galactosidase), whose expression is driven by three different GAL4-binding promoters (Figure 2). All of the reporter genes are stably integrated into the genome of the Y2HGold reporter strain. This strategic combination of four reporters virtually eliminates false positives, especially those arising from spurious GAL4 promoter-binding prey proteins, which might bind one promoter sequence but not all three.

Superior to nutritional selection alone

The use of auxotrophic reporters alone for Y2H screening often requires optimization steps, especially in the case of HIS3-based selection, to ensure that the growth conditions are sufficiently selective. The leakiness of HIS3 selection necessitates the use of 3-AT (a competitive inhibitor of the His3 protein), which must be titrated and included in the growth medium to suppress the growth of background colonies. With AbA selection and the two nutritional markers of Matchmaker Gold, growth selection is sufficiently stringent so as to make the use of 3-AT unnecessary.

Mate & Plate libraries and screening

Another major advantage of Matchmaker Gold is that we’ve replaced cumbersome library-scale yeast transformation with an easy Mate & Plate strategy that consists of combining the two haploid yeast strains that independently express the bait and prey fusion proteins (Figure 3). Y2HGold is a MATα strain which harbors the four integrated reporter genes and is transformed with a pGBKT7 plasmid to express the bait protein. The library prey strain, Y187, is a MATα strain and the ideal mating partner for Y2HGold.

We offer a variety of pretransformed Mate and Plate libraries in Y187 (see technical note). Alternatively, users can either create and transform their own library using the Make Your Own “Mate & Plate” Library System (see technical note), or transform a plasmid expressing a user-selected prey fusion protein. Combining the Y2HGold and Y187 strains for mating is very easy and results in diploid yeast that coexpress the desired combinations of the bait and prey proteins.

High numbers of confirmed positive clones

Due to the strong selective power of AbA resistance, first-round, low stringency screening selects only for blue, AbA-resistant colonies. Two-thirds of these putative positive clones are later confirmed by high-stringency screening, which selects for all four reporters. As a result, we now recommend low-stringency screening to generate a large pool of colonies, followed by high-stringency verification. This leads to more genuine positives and fewer false positives (Figure 4).

Aureobasidin A resistance is highly selective

To demonstrate the highly selective properties of AbA resistance, AbA was titrated against three diploid Y2HGold clones that each expressed different bait/prey protein pairs (Figure 5). A negative control strain that coexpressed a noninteracting protein pair (BD-POU_mOct4 bait and an unmodified GAL4 AD protein, AD-null), was completely unable to grow in the presence of AbA concentrations above 40 ng/ml. When the BD-POU_mOct4 bait was paired with a putatively interacting prey protein (AD-E2I), colonies were able to grow in the presence of 70–100 ng/ml AbA. This result suggests that these proteins interact well enough to activate AUR1-C expression and allow colony growth on AbA medium. In a positive control, the strongly interacting protein pair of DNA-BD-p53 and AD-SV40 large T antigen enabled the vast majority of colonies to grow in the presence of 100 ng/ml AbA—the highest concentration tested, indicating a very high level of AUR1-C expression.

Our highly stringent four-reporter Matchmaker Gold Yeast Two-Hybrid System and our wide selection of tissue-specific, normalized, and universal Mate & Plate libraries offer the most convenient and advanced Y2H screening tools available. You can even construct your own library with the Make Your Own “Mate & Plate” Library System. Library screens can now be accomplished in less time and with greater confidence than ever before.

Chien, C. T., Bartel, P. L., Sternglanz, R. & Fields, S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. U. S. A. 88, 9578–82 (1991).

Fields, S. & Song, O. A novel genetic system to detect protein-protein interactions. Nature 340, 245–6 (1989).

Takesako, K. et al. Aureobasidins, new antifungal antibiotics. Taxonomy, fermentation, isolation, and properties. J. Antibiot. (Tokyo). 44, 919–24 (1991).