Classification of in vivo methods used to study protein-protein interactions

There are many ways to classify in vivo methods that are used to study protein-protein interactions. If the focus is particularly on the detection and quantification of the interaction it can be classified using the properties of the reporter system used by the respective methods.

All methods presented here are based on a reporter system consisting of two parts. The first part is linked to one of the proteins of interest sometimes called the “bait” protein and the second part is the other protein of interest sometimes called the “prey” protein. When both proteins interact with each other the two parts of the reporter system are brought into proximity and have a clearly detectable effect. The terms “bait” and “prey” are normally used if there is a protein of interest and the objective is to “hunt” for interacting partners. In many cases both partners are already known, and the objective is to study the interaction in more detail (for example, its regulation).

The methods mentioned below are discussed in more detail in Xing et al. 2016.

Methods according to the functionality of the reporter system

An important difference is whether the two parts of the reporter system are fully functional by themselves or not.


Protein fragment Complementation Assays (PCA)

In the protein fragment Complementation Assays (PCA), the reporter is a single protein that has been cleaved into two fragments. The fragments by themselves are not functional but the protein can reconstitute itself when both fragments are brought into proximity of each other will restore its functionality. For example, in the split luciferase complementation test the luciferase molecule is split into two fragments and neither can produce light, but if both fragments are close enough the luciferase is reconstituted and can produce light again. Examples of PCA methods are the split ubiquitin system and the split luciferase complementation assay.

Two-hybrid assays

In two-hybrid assays, each part of the reporter system is a protein or protein domain that is fully functional, but a specific, measurable effect is produced when both parts are close enough. For example, in yeast two-hybrid assays, both the DNA-binding domain that is linked to one of the proteins of interest and the transactivation domain that is linked to the other protein are independently stable and functional, but the reporter gene is only expressed if they are in close proximity to each other. Similarly, both fluorescent proteins used in a FRET assay are fluorescent, but the acceptor protein emits light only if there is interaction between the proteins of interest.

Methods according to the detected signal

Another important classification is the type of signal generated by the reporter which is used to detect the interaction. This affects the instrumentation required for detection, the possibilities it provides for accurate quantification and suitability for high-throughput analysis.

Methods based on gene expression

In this type of method, if both proteins of interest interact the combination of the DNA-binding domain and the transactivation domain drives the expression of the reporter. Reporters are normally having two possible types: prototrophic markers, such as HIS3 or ADE2 enabling survival on depleted growth medium or chromogenic enzymes such as ß-galactosidase, which can be used for the selection via blue/white coloring of the colonies. Both the yeast two-hybrid assay and the split-ubiquitin system (see below) are based on gene expression.

This type of reporter is not capable of following dynamic changes in the interaction and can only be quantified by absorption by using a chromogenic enzyme. Such reporters can be used to evaluate large numbers of binary interactions by mixing two haploid mating types that have been transformed with different sets of proteins of interest. This is very useful for large scale screening of protein-protein interactions.

In the classical version of this method the N-terminal DNA-binding domain of the transcriptional activator GAL4 is bound to one protein of interest and the C-terminal transactivation domain to the other one. When both proteins interact, their association produces a chimeric transcription factor that activates gene expression downstream of the upstream activation sequence GAL1 (Fields and Song 1989). Further split probes have been successfully established. The yeast two-hybrid assay is easy to perform but can only be used to study interactions between proteins with nuclear localization.

This system is similar in concept to the yeast two-hybrid system and is just as easy to implement. However, it has the additional advantage that it can be applied to virtually any protein, not only to nuclear proteins. As explained above, the split-ubiquitin system is a kind of PCA: the protein ubiquitin is split into two non-functional fragments, each of which is fused to one member of the protein pair of interest. When both fragments interact, ubiquitin reassembles and becomes functional. This promotes cleavage via the ubiquitin-specific protease. In the classical version, this cleavage leads to the release of a transcription factor, that then activates the expression of the reporter gene.