The yeast two-hybrid nuclear system includes many parts of the GAL 4 system, LexA system and ProQuest system. Among them, the GAL 4 system skillfully uses the properties of the GAL 4 transcription factor in S. cerevisiae. The reporter gene design embedded in this system effectively reduces the chance of false positive results. The GAL 4 system is suitable for screening of interacting proteins, identification / validation of protein interactions. The BD of the LexA system is composed of an intact prokaryotic protein LexA, while the AD consists of a B42 protein of 88 amino acid residues from E. coli, and this combination may be applicable for some specific protein interaction studies. The LexA system may have advantages over the GAL 4 system in some specific cases, due to its unique BD and AD combinations. ProQuest The system uses advanced Gateway construction technology to show excellent recombination efficiency and cloning performance. This system performs well in the clonal expression of large segments of genes, thus being able to meet the more complex gene architecture challenges in protein interaction studies. The ProQuest system contains three different reporter genes (HIS 3, URA 3, and lacZ) to reduce false-positive results. ProQuest With its excellent cloning and expression efficiency, it occupies an important position in high-throughput screening and proteomics research.

The analysis of the original results has significantly improved the data quality of the protein interaction set. To obtain high-quality Y2H data, it is very important to consider at least the following three parameters. Auto-activation is the background self-activation intensity of the measured bait. Ideally, activation (i. e. sterile colony growth should not be observed in non-interacting pairs or vehicle controls). Viscous prey, for each prey, calculates the number of decoys (prey number) with different interactions; prey interacting with a large number of decoys is non-specific ("sticky" prey) and therefore may not have biological relevance. The threshold also depends on the nature of the bait and the number of bait screened: if a large class of related proteins is screened, many of them are expected to find the same prey. As a general criterion, the number of baits that interact with a particular prey should not be greater than 5-10% of the number in an unbiased bait set or genome-wide screen. Furthermore, more sophisticated statistical evaluations of the raw data can be performed, using the Logistic regression approach, which uses statistical and topological descriptors to predict the biological relevance of protein-protein interactions obtained from high-throughput screens and integrate known and predicted interactions from various sources.

The Y2H system was developed by Fields and Song. It utilizes the yeast GAL 4 protein, which contains the DNA binding domain and the transcription activation domain. Here, one 'decoy' protein is fused to the 'Gal 4' DNA binding domain, while another putative interaction partner, 'prey', is fused to the GAL 4 activation domain. When both recombinant proteins are expressed in the appropriate S. cerevisiae strain, the bait and prey proteins can physically interact. This behavior remodeling functional transcription factors and leads to expression of the reporter gene. These reporters can be auxotrophic markers, antibiotic resistance conveyor, or color indicators, which are often used in combination to improve the accuracy of the system. To identify new interaction partners, a library screening approach was used to identify pairwise interactions between the identified target protein (bait) and the interaction partner (potential prey) present in the cDNA library.Y2H is a common method for probing interactions between proteins. Compared to biochemical in vitro methods (e. g., mass spectrometry, ITC, or SPR) that require protein purification, Y2H is based on genetic testing. It relies on intracellular expression of two target proteins (commonly referred to as Bait and Prey). The yeast two-hybrid nuclear system includes the GAL 4 system, the LexA system, and the ProQuest system.

Although Y2H screening has always been one of the most effective methods to detect binary protein-protein interactions, one limitation of this technique is the high incidence of false-negative interactions (undetected true interactions), at about 70-90%. As with any other analysis system, the two-hybrid system has the potential to produce false positives. False positives may be either technical or biological in nature."Technical" false positive it is not based on the assembly of the two hybrid proteins. Biological false positives involve true two-hybrid interactions with no physiological relevance. These include partners that can physically interact but are never close to each other in cells due to having different subcellular localization or expression at different times in the life cycle. Often, such false positives are associated with a bait protein that acts as a transcriptional activator.Some bait or prey proteins may affect general colony viability, thereby enhancing the ability of cells to grow and activate reporter genes under selective conditions. Mutations or other stochastic events of an unknown nature can also be used as possible explanations.Numerous programs have been developed to identify or avoid false positives, including the use of multiple reporter genes, independent specificity test methods.Or simply retest the interactions in pairwise Y2H assays to ensure that the interactions are reproducible.

Yeast two-hybrid (Y2H) determination is a powerful technique to identify protein-protein interactions. However, the most common auxotrophic markers of Y2H reporters take several days to produce data, and a subjective evaluation of semi-quantitative data is required to identify interactions. The initial Y2H assay utilized a lacZ reporter expressed under the control of the Gal 4 reactive GAL 1 promoter, which allows PPI detection by blue formation in the presence of the β -galactosidase substrate. These strains facilitate the screening of cDNA libraries to identify novel interactions with target proteins and by requiring expression of two or more auxotrophic reporters, increasing the number of 3-AT (competitive inhibitor of His3 in Y2H selection medium) or changing the number of LexA operators upstream of the reporter. The β -galactosidase assay has been adapted to higher throughput forms with modified to improve sensitivity. As an alternative, GFP has been investigated as a quantifiable yeast reporter gene. The feasibility of using GFP as a reporter gene was first demonstrated by the plasmid-encoded GFP, and subsequent studies integrated the yeast codon-optimized enhanced GFP (yE GF P) into the yeast genome. Secreted night firefly luciferase (CLuc) resembles β -gal as a Y2H reporter under strong and weak interactions.