Two-hybrid screening has been developed over the past decade to enable the identification of protein–protein and protein–DNA interactions along with characterization of their interactions and their manipulation.
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Interaction between proteins plays a vital role in several biological mechanisms. The yeast two-hybrid system is used to identify a large number of protein interactions in vivo. The standard properties of eukaryotic transcription factors such as GAL4 have promoted this technique.
Transcription activators possess at least two discrete functional domains directing the transcription factors to bind with a promoter DNA sequence and activating the transcription process.
The two-hybrid system exploits the fact that the DNA-binding domain of GAL4 activates the transcription only by physical interaction, but is not crucial to associate with an activating domain.
Advantages of the two-hybrid system
The two-hybrid system is popular due to its flexibility and rapid isolation of interacting proteins.
As the technique is used to identify protein interactions in a living yeast cell, it offers a number of advantages, including protein purification and antibody development at low cost, as well as a less time consuming method of detecting of novel interacting proteins, compared with conventional biochemical and genetic methods.
It is an in vivo technique involving yeast as a host test tube. Yeast cells represent a higher eukaryotic form, which exhibit the reality closer than in vitro approaches or bacterial expression techniques.
Compared to classical biochemical approaches, where large quantities of highly purified proteins or antibodies are required, only a cDNA or a specific gene of interest is needed in two-hybrid system.
The two-hybrid system is also used to analyze known interactions by either identifying critical residues for interaction or by functional characterization of the complete subdomain. From this technique, affinities of the protein interactions are also determined by performing semi-quantitative analysis.
The most significant features of the two-hybrid system are the two-pronged technique of identifying an interacting protein and cloning of the gene.
The technique is also represented as functional screens because that helps in identifying the function of the protein when interacting with already known protein function.
Drawbacks of the two-hybrid system
Even though the two-hybrid system has many positive approaches in molecular biology, it cannot provide a solution for all of the protein–protein issues.
Since 1989, the two-hybrid approach has evolved with new developments to increase its applicability.
One of the most significant drawbacks of this technique is determining whether the specific protein of interest can initiate transcription. The approach can be successful only when the protein activates the transcription on its own.
The other concern of the technique is the wide use of chimeras.
The synthetic fusion proteins always pose a possible risk of altering the actual symmetrical arrangement of the bait or prey and, therefore, modifying its functionalities. This might also result in limited activity or in the inaccessibility to binding sites.
One of the major disadvantages is the use of Saccharomyces cerevisiae as the host cell; the protein of interest must be able to fold correctly with stability within the yeast cell.
Another downside is that some protein interactions depend on post-translational modifications that may not occur or occur unsuitably in yeast.
The modifications include glycosylation, formation of disulfide bonds, and phosphorylation. However, a few new two-hybrid systems are trying to overcome this difficulty by co-expressing the enzymes that cause posttranslational changes.
There is a possibility for disadvantage in the case of a protein or extracellular protein that consists of powerful targeting signals when fusion proteins target the yeast nucleus in the two-hybrid system. A good depiction of cDNA libraries is required to screen it.
In yeast, some proteins may become toxic at the time gene encoding protein is expressed.
There are several proteins such as cyclins or homeobox gene products that are toxic to the yeast cell nucleus. This problem can be avoided by using an inducible promoter. This prevents the crucial yeast proteins involved in DNA binding domain or activation domain getting proteolyzed by other proteins transfected into the yeast.
False positives and false negatives are found to be high in two-hybrid systems.
False negativity might occur when steric hindrance is caused by fused reporter proteins of yeast.
An additional reason for causing false negatives is dissimilar or absent posttranslational modifications of proteins in the yeast system when investigating protein interactions among higher eukaryotes.
During this situation, the modifying enzymes might co-express, along with the prey and bait. This coexpression is successful in identifying tyrosine-phosphorylation-dependent interactions. Even the temporary short-lived interactions might not be detected by this technique.
Additionally, the absence of complex protein modifications such as complex glycosylation in the yeast host cell emerges to be more challenging to overcome.