Recognized as a reputable protein-protein interaction assay service provider, Creative Biolabs is committed to offering comprehensive yeast two-hybrid (Y2H) solutions, one of the most standardized in vivo approaches for protein-protein and interaction screening and validation, for the discovery or validation of new interactions. Both the GAL4 Y2H system for nucleoprotein interaction screening and the DUAL Y2H system for membrane protein interaction screening are available based on our optimized Y2H platform.

Introduction to Yeast Two-hybrid

All processes in living cells virtually depend on protein-protein interactions (PPIs). Thus, molecular biology and disease research need to understand PPI networks. Yeast two-hybrid (Y2H) is an extremely powerful technique for studying protein-protein interactions and especially, can be used to search for a novel interacting partner by screening a single protein or domain against a library of other proteins. It is this latter characteristic that finding the interacting protein without any prior identity of such proteins is the strongest application of Y2H assay.

Y2H as a cogent genetic system is one of the most standardized approaches currently for mapping PPIs both at a small scale and in a high throughput manner. In Y2H screening, PPIs are detected by the activation of reporter genes which respond to a reconstituted transcription factor (TF). Y2H is commonly applied to test some "prey" proteins for interactions with a single "bait" or target protein or pool of proteins. The merit of this method is the direct identification of interacting protein pairs without downstream experiments since the prey protein has been known and does not need further confirmation.

Fig.1 A simple flowchart for a random yeast two-hybrid (Y2H) library screening service. (Creative Biolabs)

GAL4 Y2H System for Nucleoprotein Interaction Identification

Traditional Y2H system for PPI detection was developed based on the principle that the Gal4 transcription factor in the yeast Saccharomyces cerevisiae was composed of two functional domains, one is the DNA-binding domain (BD) mediating and the other is the activation domain (AD) responsible for transcription activation. DNA-BD located in the N-terminal 1-147 amino acid residues can recognize and bind to the upstream activating sequence (UAS) of the GAL4 response gene. AD located in the C-terminal 768-881 amino acid residues is responsible for initiating the downstream gene transcription of UAS by binding to other components in the transcriptionmachinery. Either DNA-BD or AD can't activate the transcription when functions separately. Only the two domains are sufficiently close in space, will they represent complete GAL4 transcription factor activity and activate the UAS downstream promoter, so that the downstream gene of the promoter can be transcribed.

Fig.2 The yeast two-hybrid system is a genetic tool used to detect interactions between two proteins. (Mehla, 2015)

DUAL Y2H System for Membrane Protein Interaction Identification

Due to the limitations of the traditional GAL4 Y2H system, which is only available for the PPI detection of nucleoproteins, the novel DUAL Y2H technology for the PPI detection of membrane proteins has been developed, expanding the application range of Y2H. Different from the GAL4 Y2H system based on the Gal4 transcription factor, the DUAL Y2H system screens the PPI based on the principle of split-ubiquitin. The wild-type ubiquitin is a highly conserved protein with Nub terminus and Cub terminus, playing important roles in the degradation of proteins. To apply for the PPI detection, the isoleucine at position 3 of ubiquitin Nub was mutated to glycine (NubI was mutated to NubG), leading to a significant reduction in the affinity and no reassociation between Nub and Cub, thereby resulting in abnormal function of ubiquitin.

Generally, the bait protein of interest and prey library/protein are fused to the Cub and NubG respectively. The NubG and Cub will be brought together into proximity once the bait and prey interact with each other, thus resulting in the reconstitution of a full-length and normal function of ubiquitin. The reconstituted ubiquitin is recognized and catalyzed by ubiquitin-specific proteases (UBPs), releasing the transcription factor, which further enters the nucleus and activates the transcription of reporter genes.

Fig.3 Main components of the membrane-based split-ubiquitin Y2H system. (Ivanusic, 2015)

Advantages and Key Features of Our Y2H Services

Creative Biolabs has completed plenty of protein-protein interaction screening or validation based on yeast hybrid systems, accumulating abundant expertise and experience in protein-protein interaction identifications. Added by rich experience and the perfect technology platform, our seasoned scientists offer one-stop or tailored Y2H services on a strictly fee-for-service basis. Both the GAL4 Y2H system and the DUAL Y2H system are available based on your needs.

• Fully customized: library construction, prey screening, target validation, or one-stop services are all available based on the client's specific demands.
• Low rate of false positives: we have extensive experience in handling Y2H systems. Also, a large number of premade Y2H cDNA libraries are available of your choice.
• High transformation efficiency: We use high-transformation efficient Y2HGold and NMY51 yeast strains, which carry in their genome three reporter-genes, HIS 3, lac Z, and MEL 1.
• Abundant experience: technicians who provide you with drug-target interaction identification services have more than 10 years of yeast two-hybrid research experience.

Other optional two-hybrid systems:

• Mammalian Two-Hybrid Service
• Bacterial Two-Hybrid (B2H) Service
• Reverse Yeast Two-Hybrid (rY2H) Service

Creative Biolabs optimized Y2H protocols. The efficiency and quality of our Y2H system have been satisfying clients with highly reproducible and reliable results. We are also skilled at other types of two-hybrid programs, such as mammalian two-hybrid and bacterial two-hybrid services, to suit worldwide scientists' needs in a wide range of biological fields. Please contact us for more details.

Published Data

• Venom Protein-Protein Interactions Detection Using Yeast Two-Hybrid System

Fig. 4 Yeast two-hybrid analysis. Colony growth on DDO medium (without leucine and tryptophan) indicates the successful binary co-transformations, while colony growth on QDO medium (without histidine, leucine, tryptophan, and adenine) shows the protein-protein interactions (PPIs). (Ying Jia, 2021)

Proteins and peptides are the main components of snake venom. In order to detect protein-protein interactions in venom, scientists used phospholipase A₂s (PLA₂s), the most common component of snake venom, as "bait" to analyze and identify the interaction between PLA₂s and 14 most common proteins in Western diamondback rattlesnake (Crotalus atrox) venom by using yeast two-hybrid system. As a result, the researchers identified the interaction between PLA₂s and itself, as well as the interaction between lysing-49 PLA₂ (Lys49 PLA₂) and the secreted protein (CRISP) rich in cysteine in the venom. In order to reveal the complex structure of Lys49 PLA₂-CRISP interaction from the structural level, they first used the computer program to simulate the 3D structure of Lys49 PLA₂ and CRISP. Then the binding mode of Lys49 PLA₂-CRISP interaction is predicted by three different docking programs. In addition, the most possible structure of Lys49 PLA₂-CRISP complex was inferred by molecular dynamics simulation.

• Unraveling Protein Interactions Using the Integrative Yeast Two Hybrid—High Throughput Sequencing Approach

Fig. 5 Bam35–B. thuringiensis Y2H interactome screening. (Ana Lechuga, 2021)

Bacillus virus Bam35 is the model Betatectivirus and member of the family Tectiviridae, and is considered to be an evolutionary link between prokaryotic and eukaryotic virus populations. In addition, betatectiviruses can infect bacteria of the Bacillus cereus group, which is widely used in industry and contains many pathogens. Here, the researchers generated and thoroughly analyzed the genomic library of Bam35-Bacillus thuringiensis model by using an integrated traditional yeast two-hybrid system and high-throughput sequencing (Y2H-HTS), and screened interactions with all viral proteins using different protein pairs. Overall, host metabolic proteins and peptidases are particularly abundant in the detected interactions, thus distinguishing the host-phage system from other reported host-phage PPIs. The method shown in this paper also shows the biological role of several Bam35 proteins with unknown functions, including membrane structural protein P25, which may be the center of the virus and play a role in host membrane modification during virion morphogenesis. This work gives researchers a better understanding of the interaction of Bam35-Bacillus thuringiensis at the molecular level.

FAQ

1. What is the yeast two-hybrid system used for?

   The yeast two-hybrid system is a molecular biology technique used to study protein-protein interactions. It involves the reconstitution of a functional transcription factor when two proteins of interest interact in the nucleus of a yeast cell. This system is particularly useful for discovering novel interactions or confirming suspected interactions between proteins, providing insights into protein functions and the pathways they are involved in.

2. How does the yeast two-hybrid system work?

   In the Y2H system, the interaction between two proteins is detected through the reconstitution of a transcriptional activator, which can then drive the expression of a reporter gene. The system utilizes two fusion constructs: one protein is fused to the DNA-binding domain (DBD) of a transcription factor, and the other protein is fused to the activation domain (AD). If the two proteins of interest interact, the DBD and AD come together to form a functional transcription factor that activates the reporter gene, leading to a detectable phenotypic change in the yeast, such as growth on selective media or color change.

3. Can the yeast two-hybrid system be used to study interactions between more than two proteins?

   Typically, the yeast two-hybrid system is designed to detect interactions between two proteins. However, modifications of the system, such as the yeast three-hybrid or even the yeast four-hybrid systems, have been developed to study interactions among multiple proteins. These systems use additional fusion proteins and reporter constructs to facilitate the detection of complex protein interactions involving more than two components, allowing researchers to explore multi-protein complexes and their dynamics in a cellular context.

4. How are false positives minimized in yeast two-hybrid assays?

   Several strategies are employed to reduce these false positives, including using multiple reporter genes to confirm the interaction, employing different yeast strains to test the robustness of the interaction, and using controls that include known interacting and non-interacting protein pairs to benchmark the results. Post-assay validation techniques such as co-immunoprecipitation or pull-down assays can also confirm the interactions detected in the yeast two-hybrid system.

5. What kinds of proteins can be studied using the yeast two-hybrid system?

   Yeast two-hybrid system is versatile and can be used to study transcription factors, kinases, membrane proteins, and small peptide ligands. It is particularly well-suited for soluble proteins that can be expressed and folded properly in the yeast nucleus. Proteins that require specific post-translational modifications or are part of large complexes may not be amenable to study in this system without specific adaptations or considerations.

6. Can the yeast two-hybrid system be used with proteins from any organism?

   Flexibility is one of the strengths of the yeast two-hybrid system. Proteins from bacteria, plants, animals, and even humans have been successfully expressed in yeast for Y2H assays. However, the key consideration is whether the protein can be properly expressed and remain stable in the yeast cells. Proteins that are toxic to yeast or those that require specific cofactors or chaperones not present in yeast might not be suitable for Y2H studies without additional engineering or support systems.

7. What are the common readouts or reporters used in yeast two-hybrid assays?

   Common reporters used in yeast two-hybrid assays include genes that allow for selection based on growth or survival, such as HIS3, ADE2, or LEU2, which confer the ability to grow in the absence of histidine, adenine, or leucine, respectively. Additionally, colorimetric reporters such as LacZ, which encodes β-galactosidase leading to blue color formation in the presence of X-gal, are frequently used. These reporters provide a straightforward means to detect and quantify the interaction of proteins by correlating with the growth or color change of the yeast colonies.

8. How is the specificity of protein interactions tested in the yeast two-hybrid system?

   To test the specificity of protein interactions in the yeast two-hybrid system, researchers often perform mutagenesis studies, where key amino acids involved in the interaction are mutated to assess their impact on the interaction strength. This helps to confirm which parts of the proteins are critical for binding. Additionally, researchers may use competition assays where a third protein is introduced to see if it can disrupt the interaction between the two primary proteins of interest. These methods help to ensure that the observed interactions are specific and not due to non-specific aggregation or overexpression artifacts.

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