7.3.1 Library-based methods for demonstrating an interaction between proteins
As well as the biochemical approaches to studying protein–protein interactions, there is a variety of qualitative methods for screening ‘libraries’ of cloned genes or gene fragments whose protein products might interact with a protein of interest. Such an approach has the advantage that the genes that encode those proteins that bind are available immediately for expression, facilitating subsequent analysis of the protein.
The two-hybrid system uses transcriptional activity as a measure of protein–protein interaction, as illustrated in Figure 51.
The ‘read-out’ from this system is expression of a so-called reporter gene, i.e. a gene whose expression is readily monitored (as, for example, enzyme activity, or fluorescence). The technique exploits the fact that the protein that activates transcription of the reporter gene consists of two domains: (1) the DNA binding domain, which targets the activator to specific genes, and (2) the activation domain, which contacts other components of the transcriptional machinery, enabling transcription to occur. Though normally these two domains would be part of the same polypeptide, transcription of the reporter gene can still occur if they are on different polypeptides but are brought together physically. The two-hybrid system entails expression, using recombinant DNA techniques, of two hybrid proteins as follows:
The DNA binding domain is fused to the bait protein, X.
The activation domain is fused to the potential binding partner, Y.
If X and Y interact, the DNA binding domain and the activation domain are brought together such that transcription of the reporter gene can occur. The two-hybrid assay is commonly performed in yeast cells containing a reporter gene. The major advantage of this system is that it allows the screening of large libraries consisting of many genes fused to the DNA encoding the activation domain.
Another widely used library-based method for detecting protein–protein interactions is phage display (Figure 52). This approach uses a virus that infects E. coli, known as a bacteriophage (or ‘phage’). Similarly to the two-hybrid system, recombinant DNA techniques are used to make a gene construct by fusing DNA encoding the proteins of interest with a gene encoding a protein that forms part of the coat of the phage. Expression of this construct produces a hybrid of the bacteriophage coat protein and the protein of interest. A coat protein is chosen for this manipulation so that the hybrid will be displayed on the surface of the mature engineered phage. Engineered phage derived from a library of such constructs can be screened for the ability to bind to a bait protein. Immobilised bait protein could be used in the same way as described for affinity chromatography (Figure 49a), to fish out any phage expressing interacting proteins. The recovered phage can then be used to infect E. coli. In this way, the phage can be replicated to increase its yield and facilitate subsequent isolation of the DNA encoding the interacting protein.