Protein-protein interaction plays an essential role in virtually every cellular process and the identification of it is a key step to understand unknown and complex protein functions. So far, Creative Biolabs has provided a bacteria-based genetic selection system, Bacterial Two-hybrid (B2H, BTH), to explore the details of protein’s interaction and achieve clients’ requirements on it.

The bacterial two-hybrid is a rapid genetic approach to detect and characterize interactions between a wide variety of bacterial, eukaryotic, or viral proteins in vivo. Similarly to the classic yeast two-hybrid (Y2H), bacterial two-hybrid is based on reconstituting the activity of a certain protein from its separate domains and a reporter gene is expressed only when the relative protein-protein interaction occurs within a test cell. It is worth noting that the phenomenon of autoactivation by baits is a less problem in bacterial systems than in the yeast. Therefore, the bacterial method provides an alternative strategy for studying proteins which cannot be assayed in Y2H because of toxicity or low expression.

The Principle of Bacterial Two-hybrid

The bacterial two-hybrid was initially devised using bacteria as a host organism in 1997. It’s a transcriptional activation-based two-hybrid approach and has become a common laboratory tool preferred in some specified circumstances.

This is a two-hybrid method in Escherichia coli where the proteins of interest are fused to two complementary fragments of the catalytic domain with Bordetella pertussis adenylate cyclase, T25, and T18, that are not active when physically separated. Interaction of these two proteins results in functional complementation between the two adenylate cyclase fragments, which leads to cAMP synthesis and in turn, can trigger the expression of several resident genes, such as lac and mal operons. Using this assay, scientists can select specific clones expressing a protein that interacts with a given target. And with the development of bacterial two-hybrid, proteins of bacterial origin can be analyzed for interactions under conditions that match their native environment more closely.

Fig.1 Principle of bacterial two-hybrid systems.

Workflow and Advantages

There are four facile steps in a bacterial two-hybrid system: construction of plasmids, transformation of microbial cells, intracellular expression of fusion proteins, and selection for reporter product. Taking E. coli as host offers potentially significant advantages over analogous two-hybrid systems:

• Allowing to screen larger libraries of proteins (>10⁸ in size)
• Ease of use, fast growth, and high transformation efficiency
• A low false positive rate
• Nuclear localization not required
• Greater potential permeability to small molecules
• More possibility of detecting proteins that are toxic when expressed in yeast
• Fewer indirect interactions affected by endogenous proteins

Moreover, the bacterial two-hybrid selection system from Creative Biolabs can not only study interactions of different proteins from both prokaryotes and eukaryotes but also apply successfully in the protein-DNA analysis, and identify antigen-specific single domain antibodies.

Other optional two-Hybrid systems:

• Yeast Two-Hybrid (Y2H) Service
• Mammalian Two-Hybrid Service
• Reverse Yeast Two-Hybrid (rY2H) Service

Published Data

• KatG-Lon Interaction Study by Bacterial Two-Hybrid (B2H) Assay

Fig. 2 Confirmation of the KatG-Lon interaction. (Perumalraja Kirthika, 2022)

In this paper, the researchers investigated the interaction between Lon protease and catalase-peroxidase (KatG) and their effects on virulence regulation and the oxidative stress response of Salmonella Typhimurium (ST). Through proteomic comparison between ST wild type and lon deletion mutants, a highly expressed KatG protein was identified, while the other five candidate proteins were significantly affected by lon deletion. Through the bacterial two-hybrid (B2H) assay, it was proven that the catalytic domain of Lon protease might interact with KatG protein, thus leading to protein hydrolysis. The evaluation of virulence gene expression in single and double mutant lon and katG mutants showed that katG was a potential positive regulator of both Salmonella pathogenicity Island-1 (SPI-1) and -2, while lon significantly affected the SPI-1 gene. ST double deletion mutants are more likely to show survival defects in macrophage-like cells. Compared with the single deletion mutant, the colonization rate of the spleen in mice was lower. The experimental results explored the previously unknown function of the interaction between Lon and KatG in the toxicity of Salmonella.

FAQ

1. What is the bacterial two-hybrid (B2H) system, and how does it work?

   The bacterial two-hybrid (B2H) system is a method used to study protein-protein interactions within a bacterial cell, typically Escherichia coli. This system is based on the reconstitution of a functional transcription factor when two proteins of interest interact. In B2H, each protein of interest is fused to one of two different domains of a transcription factor. If the proteins interact, the domains come together to form a functional transcription factor, which then activates the transcription of a reporter gene. This reporter gene is engineered to produce a detectable signal, such as antibiotic resistance or a colorimetric change, indicating the interaction of the proteins.

2. What are the advantages and limitations of using the B2H system?

   The advantages of the B2H system include its simplicity and cost-effectiveness, as it does not require the use of sophisticated equipment or eukaryotic cell culture systems. It also allows for rapid screening of large libraries of protein interactions. However, the B2H system has limitations, including the potential for false positive and false negative results. False positives can occur due to non-specific interactions between the fusion proteins or the transcriptional machinery, while false negatives can arise if the fusion disrupts the natural interaction between the proteins or if the proteins do not fold properly in the bacterial cell. Additionally, since the system is used within a bacterial context, the interactions observed may not always accurately reflect how proteins interact in eukaryotic cells or in more complex biological environments.

3. How is the bacterial two-hybrid system set up for detecting protein interactions?

   The first is to construct plasmids where the genes encoding the proteins of interest are fused to the genes encoding either the DNA-binding domain or the activation domain of a transcription factor. These plasmids are then transformed into a suitable bacterial strain that contains a reporter gene under the control of a promoter activated by the transcription factor. Once in the bacterial cell, if the proteins of interest interact, the two domains of the transcription factor are brought together, which reconstitutes a functional transcription factor that activates the reporter gene. The resulting signal, which could be enzymatic activity leading to a color change or resistance to an antibiotic, indicates a positive interaction between the studied proteins.

4. Can the B2H system be used to screen for inhibitors of protein-protein interactions?

   A bacterial two-hybrid assay is particularly useful in drug discovery and development processes where blocking a protein interaction is the therapeutic goal. In this adapted B2H assay, compounds are introduced to the bacteria harboring the B2H setup. The presence of a compound that inhibits the interaction between the target proteins will prevent the reconstitution of the functional transcription factor, thereby reducing or abolishing the reporter gene expression. Screening libraries of small molecules or peptides using this system can help identify potential inhibitors that can be further developed as drugs.

5. What are the common reporter genes used in the bacterial two-hybrid system?

   In the bacterial two-hybrid (B2H) system, common reporter genes include those encoding enzymes such as β-galactosidase (lacZ), which can be easily assayed by a colorimetric reaction that turns blue in the presence of the substrate X-gal. Another frequently used reporter is the chloramphenicol acetyltransferase (CAT) gene, which confers resistance to the antibiotic chloramphenicol, allowing for the selection of positive interactions. Additionally, the use of antibiotic resistance genes like those for ampicillin or kanamycin is common, as these allow for the growth of only those bacteria that express the reporter gene, thus indicating a positive interaction between the fusion proteins.

6. How do environmental factors affect the results of the B2H system?

   Temperature can affect protein folding and stability, potentially altering protein interactions. Most bacterial two-hybrid assays are conducted at 37 °C, which is optimal for E. coli growth but may not be ideal for all protein interactions, especially those native to organisms from different thermal environments. Additionally, the composition of the growth medium can influence protein expression levels and the background activity of the reporter gene. Variations in ionic strength, pH, and the presence of certain ions or small molecules can also affect protein-protein interactions. It is crucial to optimize these conditions during experimental setup to reduce false positives and negatives, ensuring that the observed interactions are as physiologically relevant as possible.

7. How do researchers confirm the results of a bacterial two-hybrid assay?

   After obtaining positive results from a B2H assay, researchers typically confirm these interactions through additional experiments to ensure their validity. Common follow-up techniques include co-immunoprecipitation and pull-down assays, which physically recover the protein complexes from cell lysates and verify their interaction. For further validation, biophysical methods such as surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) can quantitatively analyze the interaction dynamics. These complementary approaches help to confirm the specificity and relevance of the interactions detected in the B2H system.

8. What modifications can be made to the B2H system to improve its sensitivity and specificity?

   To enhance the sensitivity and specificity of the B2H system, several modifications can be implemented. One approach is to use different reporter genes that may be more sensitive or provide a quantitative output, such as luciferase-based reporters. Researchers can also alter the vectors and bacterial strains used to reduce background activity and improve the dynamic range of the assay. Additionally, varying the linker sequences between the fusion protein domains and the protein of interest can help in maintaining the proper folding and orientation necessary for interaction. Using multiple reporter genes or conducting the assay under different growth conditions can also help minimize false positives and false negatives, improving the reliability of the system in detecting genuine protein-protein interactions.

9. What types of protein interactions are best studied using the bacterial two-hybrid system?

   The B2H system is particularly useful for detecting direct interactions between protein pairs, making it an excellent choice for studying binary protein interactions within a simple system. It is well-suited for initial screening of unknown interactions from complex libraries or for confirming suspected interactions suggested by other studies, and it is most effective for studying interactions where the proteins do not require post-translational modifications that are absent in bacteria. Proteins that are naturally expressed in bacteria or those that remain functional when expressed in bacterial cells are typically the best candidates for B2H studies.

10. How do differences in protein expression levels affect the outcomes of B2H assays?

    Protein expression levels can significantly influence the results of B2H assays. If the proteins are expressed at unnaturally high levels, it may lead to non-specific interactions, resulting in false positives. Conversely, very low expression levels might result in false negatives, where genuine interactions are not detected because the proteins are not abundant enough to meet each other within the cell. To mitigate these issues, researchers often optimize expression levels by adjusting promoter strength or using different bacterial strains that control expression more tightly. Furthermore, employing quantitative rather than qualitative reporter systems can help discern true interactions from background noise by allowing a more precise measurement of interaction strength based on the level of reporter gene activation.

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.