The interaction of membrane proteins in biological processes is the main focus of research in the yeast two-hybrid membrane system. These interactions are indispensable for gaining insights into cell membrane structure, functional properties, signaling mechanisms, and the function of disease-related proteins. With this technology, scientists have successfully revealed key membrane protein complexes, such as the core components of the extracellular signal-regulated protein kinase (ERK) pathway. In addition, the membrane system yeast two-hybrid technology also promotes the screening and verification of new drug targets, bringing new progress to the field of drug research and development. The technology works in eukaryotic yeast cells, ensuring that proteins maintain their native fold, thus making protein-protein interactions closer to the real state of the body. Its strong detection capability can capture weaker protein interactions, which is particularly critical for discovering those with transient or low affinity interactions. By constructing a yeast bihybrid library containing numerous membrane protein fusions, the membrane system yeast two-hybrid technology enables high-throughput protein interaction screening. During the library screening, the two-hybrid system directly obtained the gene sequence encoding interacting proteins, thus avoiding the tedious protein extraction and purification steps in other in vitro protein interaction detection methods.TEK Biotech has rich experience in yeast two-hybrid project, which can help customers better complete the exploration of the relationship between protein molecules.

The Y2H system uses the GAL 4 protein in yeast, which is composed of two parts: the DNA binding domain and the transcriptional activation domain. In this system, the "bait" protein is fused to the DNA binding domain of GAL 4, while another potential interacting object, the-"prey" protein, is fused to the activation domain of GAL 4. When the two recombinant proteins are expressed in specific strains of S. cerevisiae, the bait is capable of physically interaction with the prey proteins. This interaction is able to reconstruct functional transcription factors, which subsequently initiate reporter expression. These reporters are widely used, including auxotrophic markers, antibiotic resistance markers, and color indicators, which are often used in combination to enhance the accuracy of the system. The Y2H technology has become a common means to probe protein-protein interactions. Compared to biochemical in vitro methods (e. g., mass spectrometry, ITC or SPR), Y2H is based on genetic testing. It relies on fusions that express the two target proteins in cellular cells (often called Bait and Prey, respectively). DUALmembrane The system contains the following components: First, the isolated ubiquitin system, in which ubiquitin is divided into N (NubI) and C (Cub) for expression, and the Cub part is usually fused with LexA protein or other types of transcription factors that can initiate reporter gene expression in the nucleus. Second, the fusion expression of membrane proteins, in the DUALmembrane system, fusion expression vectors of membrane proteins and soluble proteins are constructed and linked to the N-and C-terminal parts of ubiquitin, respectively. Finally, when two fusion proteins interact, they close the N terminus to the C terminus to reorganize it into a complete ubiquitin structure, a process that would trigger a series of signaling events that ultimately induce expression of the reporter gene.

The HIS 3 gene encodes a histidine synthase that allows yeast cells to grow in a histidine-free environment. Using HIS 3 as a reporter gene, yeast cells that regain the function of HIS 3 expression by protein interactions can be identified. LacZ is another common reporter gene with a product of β -galactosidase. Expression of lacZ initiates the generation of blue products when an β -galactosidase substrate is present, facilitating the intuitive detection of protein – protein interactions. The ADE 2 gene encodes a key enzyme in the adenine synthesis pathway, similarly allowing yeast cells to survive on an adenine-deficient medium. ADE 2 can also be used as a reporter gene for probing protein-protein interactions. The metallothionein encoded by the AUR 1-C gene is resistant to arsenate. Use of AUR 1-C as a reporter gene can select for yeast cells expressing AUR 1-C due to protein interaction and subsequently obtaining arsenate resistance. The MET 25 gene encodes a methionine synthetase, allowing yeast cells to grow on a methionine-deficient medium. Moreover, MET 25 could also serve as a reporter gene for Y2H screening. GFP (green fluorescent protein) is a protein with spontaneous fluorescence characteristics and is often used to monitor the expression and localization of proteins in real time. In the Y2H system, GFP can also be used to quantify the strength of protein – protein interactions. In addition to GFP, other fluorescent proteins such as red fluorescent protein (RFP) and yellow fluorescent protein (YFP) can also be used as effective reporter genes for the Y2H system.

Analysis of the raw results significantly optimized the quality of the protein interaction dataset. There are three key factors in pursuing high-quality Y2H data.First of all, we should pay attention to the background self-activation level of the Y2H membrane system. Ideally, the activation phenomenon (i. e. sterile colony growth should not occur in non-interacting pairs or carrier control). Secondly, for each prey, the number of interactions with different baitres needs to be calculated. If the prey interacts with a large number of decoys, it may be non-specific ("sticky" prey), which may lack biological significance. Moreover, the threshold is influenced by the bait characteristics and the screening size: if the screen covers a large group of related proteins, many proteins are expected to find the same prey. In general, the proportion of decoys interacting with a particular prey should not exceed 5%- 10% in an unbiased decoy set or in a genome-wide screen. Moreover, more sophisticated statistical assessment methods can be used to combine statistics with topological descriptors to predict the biological relevance of protein-protein interactions in high-throughput screens and to integrate known and predicted interaction information from different channels.TEK Biotech is deeply engaged in the yeast platform to help clients study the relationship between protein molecules.

Specific membrane proteins cannot perform precise folding and post-translational modification steps correctly, but it is difficult to fully simulate these steps in yeast cells. In order to be closer to the native conditions, the yeast expression system can be optimized, such as regulating the culture environment, introducing auxiliary proteins and other means, and strive to simulate the folding and modification process of proteins. The intricate interactions between membrane proteins may reduce the screening efficiency and lead to a rise in false positive results. Similar to other analytical systems, the two-hybrid system also has the possibility of producing false positives. These false positives may originate from technical factors or biological factors. Technical-level false positives are not based on the actual assembly of the two hybrid proteins. Biological false positives refer to those cases in which real two-hybrid interactions occur, but have no practical significance under physiological conditions. Such cases include pairs of proteins that physically interact but are never truly close within cells because of different subcellular localization or expression patterns at different stages of the cell life cycle. Moreover, some bait or prey proteins may have an impact on the overall colony viability of the colony, which in turn enhances the ability of cells to grow in a selective environment and the efficiency of reporter activation. To identify or circumvent these false positives, multiple strategies have been developed, for example with multiple reporter genes and independent specific validation methods. Therefore, it is crucial to adopt more rigorous screening criteria, such as a double reporter system (e. g., lacZ and HIS 3), and the addition of negative control groups and a variety of protein tags to reduce the false positive rate. At the same time, with the help of advanced bioinformatics technology, the screening results can be further analyzed and verified. It is noteworthy that not all membrane proteins are smoothly compatible with yeast two-hybrid systems, especially structurally or function-specialized membrane proteins. In this regard, membrane proteins can be modified or optimized, such as adjusting their sequence or structure through genetic engineering means to enhance their systematic adaptability. In addition, exploring other protein interaction screening techniques, such as co-precipitation technology and fluorescence resonance energy transfer method, is also a feasible path.