A key part of gene functional analysis and potential drug target discovery is an understanding of how proteins interact within the cell. The two-hybrid system is a very powerful molecular genetic tool which investigates protein-protein interactions in vivo. This system can be used to study the interaction between two proteins which are expected to interact or find proteins (prey) that interact with a protein you already have (bait).

In Creative Biolabs, we can provide many types of two hybrid services, such as yeast two-hybrid (Y2H), mammalian two-hybrid (M2H), bacterial two-hybrid (B2H) or reverse yeast two-hybrid (rYTH), for global clients to identify the role of protein-protein interactions in biological systems. By using the method offered by us, scientists are able to explain structure-function relationships among proteins and mediate the biological function of interactors for therapeutic purposes, as well as to establish large-scale protein interaction maps. Additionally, as these techniques can be applied in drug discovery and development programs, they also could be new opportunities for biomedical research. We offer services on a strictly fee-for-service basis and several major two-hybrid services are introduced as follows:

Yeast Two-hybrid (Y2H)

It is acknowledged that Y2H is a greatly powerful technique for studying protein-protein interactions. In the Y2H assay, the 'bait' and 'hunter' plasmids are introduced into yeast cells by transfection. In this process, the plasma membrane is disrupted to yield holes, through which the plasmids can enter. Once transfection has occurred, cells containing both plasmids are selected for by growing cells on minimal media. Only cells containing both plasmids have both genes encoding for missing nutrients, and consequently, are the only cells that will survive.

Fig.1 Yeast two-hybrid system.

Mammalian Two-hybrid (M2H)

The principle of M2H is analogous to the better-known Y2H. The M2H technology relies on the mammalian background in which they operate and complement the established Y2H approach. This assay opens a new avenue to study the dynamics of mammalian protein interaction networks, including the temporal, spatial and functional modulation of proteins associations.

Fig.2 Mammalian two-hybrid layout.

Bacterial two-hybrid (B2H)

The B2H assay is based on the reconstitution of adenylate cyclase in Escherichia coli that was described 14 years ago. For microbiologists, it is indeed a practical and useful alternative to the use of the widely spread Y2H system for testing protein-protein interactions.

Fig.3 Bacterial two-hybrid protocol.

Reverse Yeast Two-hybrid (rY2H)

rY2H is an upside-down version of Y2H, in which protein interaction is lethal or toxic for the yeast cells. Yeast strain expressing URA3 gene can’t grow, so URA3 is usually chosen for negative selection.

Creative Biolabs has professional teams and labs for two-hybrid services and the efficiency and quality of our identifying and screening platforms have always been satisfying clients with highly reproducible and reliable results. Except that, our two-hybrid techniques have other predominant advantages as well.

• Low Rate of False Positives
  We have extensive experience in handling two-hybrid systems. Meanwhile, a large number of premade cDNA libraries are available of your choice.
• Optimized Protocols
  We optimized classical two-hybrid protocols and provide a specific customized scheme for customers according to their different purposes.
• Experienced Technical Staff
  We have more than 10 years of experience in two-hybrid research work and operational skills in experiments.
• Step by Step Cost-Effectiveness
  If the bait gene is toxic or self-active in the test, the customer only need pay our setup fee.

Published Data

• Two-hybrid System Reveals PPIs within H. pylori NIF Fe-S Maturation System

Fig. 4 Picture of a gel showing interactions between recombinant ApbC and Nfu proteins. (Stéphane L. Benoit, 2021)

Iron-sulfur (Fe-S) proteins play an important role in all organisms. Helicobacter pylori, the pathogen of gastropathy, relies entirely on NIF system for biosynthesis and delivery of Fe-S clusters. The components previously characterized include two essential proteins NifS and NifU, and a dispensable Fe-S carrier Nfu. Among the 38 proteins predicted to coordinate Fe-S clusters, the bacterial two-hybrid system was used to identify protein-protein interactions, focusing on two proteins, HP0207 and HP0277. It was found that ApbC interacts with 30 proteins, and changes in the conserved ATPase motif in ApbC significantly reduce the number of protein-protein interactions, indicating that some ApbC-target protein interactions require ATpase activity. FdxA has been confirmed to interact with 21 proteins, but no interaction between NifU and FdxA has been detected. A 51 kDa ApbC-Nfu heterodimer complex was identified by cross-linking studies. The attempt to generate apbC chromosome deletion mutant in H. pylori was not successful, indirectly indicating that the hp0207 gene is necessary. Overall, these results show that both ApbC and FdxA are important participants in the H. pylori NIF maturation system.

• Using Two-Hybrid Assay to Study Protein Interactions within Secretory Pathway

Fig. 5 The Golgi two-hybrid assay is based on the modularity of the mannosyltransferase Och1p. (Danielle H. Dube, 2010)

Because strong activation can lead to false positive, it is challenging to use transcription-based two-hybrid assays to study the interaction of transcriptional activators. Here, the researchers reported the development of Golgi two-hybrid (G2H) method for investigating protein-protein interactions in Golgi. G2H relies on protein-protein interactions in extracellular reported secretory pathways via cell surface glycosylation. Cells with interacting protein pairs were identified by selective phenotypes related to Och1p activity and appropriate cell wall formation: cells with interacting proteins grew under selective conditions and exhibited weak agglutinin binding by flow cytometry, while cells lacking interacting proteins showed growth retardation and strong agglutinin binding capacity. Using this assay, the researchers examined the interaction between the transcription factor MyoD and its binding partner Id2. In addition, they used G2H to detect the interactions between the Gal4p activation domain and various binding partners. Finally, selective conditions are used to enrich cells that encode interacting chaperones. G2H detection of protein-protein interactions that cannot be recognized by traditional two-hybrid methods should be widely used to detect previously inaccessible subsets of interaction groups, including transcriptional activators and proteins transported through secretory pathways.

FAQ

1. What is the principle behind the yeast two-hybrid system for detecting protein-protein interactions?

   The two-hybrid system is a genetic tool used to detect protein-protein interactions by exploiting the modular nature of transcription factors in yeast. This method involves splitting a transcription factor into two separate domains: a DNA-binding domain (DBD) and an activation domain (AD). These domains are functionally inactive when apart. In the two-hybrid system, one protein of interest (bait) is fused to the DBD and another protein (prey) is fused to the AD. If the bait and prey proteins interact within the yeast nucleus, they bind to the DBD and AD close enough to reconstitute a functional transcription factor, triggering the transcription of a reporter gene. This results in a detectable phenotype, such as growth on selective media or color change, indicating interaction between the studied proteins.

2. What are the common limitations of the yeast two-hybrid system in studying protein-protein interactions?

   False Positives: Some interactions detected may be artifactual, resulting from the overexpression of fusion proteins in the yeast nucleus, where they might not normally localize.
   False Negatives: True interactions might be missed if the proteins do not fold correctly in the yeast nucleus, if they require post-translational modifications absent in yeast, or if the interaction is transient and not stable under assay conditions.
   Context Dependency: The interaction detected in yeast may not necessarily reflect an interaction under physiological conditions in human cells, as the cellular context and presence of other interacting molecules can influence PPIs.

3. What are the main applications of the two-hybrid system in biological research?

   The two-hybrid system is extensively used in biological research to map protein-protein interaction networks and understand the functional organization of the proteome.
   Identifying Novel Interactors: Researchers use the system to identify unknown partners of a known protein, which can reveal new aspects of cellular function and regulatory mechanisms.
   Mapping Interaction Networks: By systematically testing combinations of proteins, scientists can construct interaction networks that provide insights into cellular pathways and processes.
   Functional Characterization: Interactions identified via the Two-Hybrid System can be further analyzed to determine the biological significance of these interactions in processes like signal transduction, gene regulation, and cellular metabolism.
   Pathogenesis Study: Understanding how protein interactions change in disease states can help identify potential targets for therapeutic intervention, especially in diseases where protein misinteraction or malfunction is a known factor.

4. How does the choice of reporter genes in the yeast two-hybrid system affect the assay outcomes?

   The choice of reporter genes in the two-hybrid system is crucial as it directly impacts the sensitivity and specificity of the assay. Common reporter genes include:
   LacZ: Produce β-galactosidase, which catalyzes the hydrolysis of X-gal, leading to the formation of a blue product. This colorimetric readout is straightforward but can sometimes yield high background levels, which affects the assay's specificity.
   HIS3: Allow yeast to grow without histidine, making it suitable for selection-based assays. This reporter is highly sensitive and can detect even weak interactions, but may also be prone to false positives without stringent control conditions.
   ADE2 or LEU2: Similar to HIS3, these reporters allow growth in media lacking adenine or leucine, respectively. They are useful for constructing more stringent selection systems that reduce the likelihood of false positives.

5. What are the limitations of the two-hybrid system in studying protein interactions involving post-translational modifications?

   Lack of Relevant Cellular Machinery: Yeast cells, commonly used in the two-hybrid system, may lack the specific enzymes required for certain PTMs that occur in mammalian cells. This absence can prevent the proper folding or function of the protein, or the interaction itself if it is contingent on a specific modification.
   PTM-Specific Interactions: Some interactions are dependent on phosphorylation, ubiquitination, or other modifications that are either absent or not accurately replicated in yeast. This can lead to false negatives where the interaction is overlooked because the critical modification is missing.
   Artificial Localization: The fusion of proteins to the DNA-binding or activation domains required for the Two-Hybrid assay might alter their native localization and modification patterns, affecting the physiological relevance of the detected interactions.

6. Can the two-hybrid system be used to screen for interaction inhibitors?

   Yes, the two-hybrid system can be adapted to screen for inhibitors of protein-protein interactions, which is particularly valuable in drug discovery. This adaptation is known as the Reverse two-hybrid system. In this setup, compounds are tested for their ability to disrupt a known interaction between two proteins. A disruption leads to the failure to activate the reporter gene, resulting in no growth or a change in the phenotype of the yeast cells. This method allows researchers to identify small molecules or peptides that can interfere with specific interactions, potentially leading to the development of new therapeutic agents that target protein-protein interaction networks implicated in various diseases.

7. What improvements have been made to the two-hybrid system to increase its utility?

   Gateway Cloning Technology: This allows for the rapid transfer of DNA sequences encoding proteins of interest into various vector systems compatible with the two-hybrid assay, speeding up the process of assay setup.
   Normalized Libraries: Improvements in library preparation, such as normalization to equalize the representation of prey proteins, enhance the chances of identifying true interactions by reducing the overrepresentation of abundant proteins.
   Automated Screening: Automation of the screening process, including robotic handling of yeast cultures and automated detection of reporter gene activity, has significantly increased throughput and reproducibility.
   Integration with Omics Data: Combining two-hybrid screening data with genomics, proteomics, and transcriptomics data helps to validate and contextualize the interactions in biological pathways and networks, providing a more comprehensive understanding of cellular functions.

Resources

Use the resources in our library to help you understand your options and make critical decisions for your study.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.