Yeast 2-Hybrid Systems

You can use the yeast two-hybrid system to study interactions between two known proteins, or you can use it to find proteins (and their corresponding genes) that interact with a single protein of interest. In the latter type of experiment, the gene encoding your known protein is typically referred to as the bait. The gene(s) that encode the protein(s) that interact with your bait are referred to as the prey. Though the terminology may sound simple, it is not always easy for new users to get started with the system.

A common complaint about the system is that it can yield a high number of false positives. Jens Boettcher of MoBiTec says that the identification of these false positive clones is especially challenging for newcomers. “It requires some experiences with yeast to distinguish between real positive clones and false positive clones,” Boettcher said. “The selection by aminoacid auxotrophy is not that stringent and the handling of 3-at (against the leaky his-auxotrophy) is sometimes difficult (temperature sensitive).”

However, Daniel Auerbach, CSO of Dualsystems Biotech, denies that the yeast two-hybrid system delivers too many false positives. “Most biological assays inherently do so—it is just that in yeast 2-hybrid these false positives are immediately obvious and can be easily identified, making their presence obvious. Doing a biochemical pulldown experiment, you usually do not realize the amount of false positives because you don’t immediately see them. This doesn’t help you though, because you will start downstream work with those proteins and it may take you quite a while before you realize that you are working on an artifact. The yeast 2-hybrid system helps you there because false positives can be identified quickly and can be sorted out. Furthermore, the more carefully a yeast 2-hybrid assay is optimized before going into the actual screen, the fewer false positives will come out. Many people do not realize that they are not careful enough in the first steps of the assay because they are so eager to get to the actual screening process. The result is usually a fairly unspecific screen with false positives.”

This article aims to cover some of the products and services offered to help you navigate your yeast two-hybrid experiments around the pitfalls and false positives.

MoBiTec offers, among other classical yeast two-hybrid kits, their Grow ‘n Glow ACE1 Yeast Two Hybrid System. Features of this kit include the selection for clones, according to Boettcher. “An additional beta-gal-test is no longer necessary; positive clones will be marked by the GFP-reporter gene,” Boettcher said; in addition, “the number of false positives is reduced by stringent selection for copper-resistance.” The MoBiTec system uses the ACE1 protein rather than the Gal4 protein, and GFP fluorescence as a reporter. ACE1 is a copper-inducible transcription factor, which gives you greater selection control, potentially reducing false positives. Altering the copper concentration can also allow you to select differing strengths of protein-protein interactions.

According to Phillip Gray, senior scientist in research and development at Invitrogen, there are many features of the ProQuest two-hybrid system that make it an attractive product. “Our competitor's system uses high copy number vectors, which may lead to an increased number of false positives.” Invitrogen uses low copy-number vectors (ARS/CEN) to mitigate this complication. Three different reporter genes (HIS3, URA3, and lacZ) with independent promoter regions also help to reduce false positives. “In addition,” says Gray, “the use of the counter-selectable reporter URA3 is unique to ProQuest. URA3 serves as a double reporter, where a positive interaction facilitates growth under uracil selection, but results in toxicity in the presence of 5-FOA. Likewise, a negative interaction will not grow under uracil selection but will grow in the presence of 5-FOA. As a result, alleles of a particular gene that no longer interact may be selected for using the URA3 reporter in the presence of 5-FOA (reverse two-hybrid system).”

While the classical yeast two-hybrid system offered by Dualsystems Biotech is based on LexA rather than Gal4, it also includes three reporter genes (HIS3, ADE2, and lacZ) to weed out false positives. “Extensive optimization of the sensitivity of each reporter gene has resulted in a reporter strain which is at the same time quite stringent (meaning fewer false positives) and yet able to pick up weak interactions,” says Auerbach. “We believe this is a major advantage when compared to other available reporter strains, which are either very stringent or generate too many false positives.”

Auerbach explained another unique feature of Dualsystems’ products—they offer “bait” construction in two different configurations: “Either the classical configuration (LexA-BAIT), where the DNA binding domain is situated at the N-terminus of the protein of interest, or an alternative C-terminal configuration (BAIT-LexA), where the DNA binding domain is located at the C-terminus of the protein of interest. This has several advantages, for example when an N-terminal fusion results in an unstable fusion protein, or when a fusion to the N-terminus of the protein of interest prevents its interaction with a protein partner.” Dualsystems offers screening services (see below), but also sells their classical yeast two-hybrid system as a kit, which includes both bait vectors, a library vector, accessory vectors and controls, the triple reporter strain NMY51 for increased screening stringency, “and a very detailed manual which will also help beginners to get started quickly,” says Auerbach.

The Matchmaker Two-Hybrid System 3, sold by Clontech, is a Gal4-based yeast two-hybrid system that also uses three different reporter genes to reduce false positives. Clontech’s Match