Two-hybrid screening

In the yeast two -hybrid system (English yeast two - hybrid system, abbreviated Y2H ) is a biochemical technique for the determination of protein -protein interactions or protein-DNA interactions.

Principle

The yeast two -hybrid system is an in vivo method for the detection of protein -protein interactions in yeast, generally the yeast Saccharomyces cerevisiae. The basis for this is a required for gene regulation protein, known as a transcription factor. In Saccharomyces cerevisiae, one normally uses the transcription factor GAL4. This has two different domains, one for binding to the DNA ( binding domain, GAL4 -BD) and one which activates transcription ( activation domain, GAL4 -AD). Although the two domains are usually located on the same polypeptide chain, these are also effective when they are brought together by two different proteins via non-covalent protein - protein interactions. For this purpose, use is made of two compatible expression vectors in yeast.

Each of the two plasmids each carrying a designed experiment for the corresponding fusion gene. This encoding in the first case for a hybrid protein consisting of the GAL4 BD and at which the amino acid sequence followed for which a potential binding partner is to be found ( bait protein bait protein). The second plasmid encodes a hybrid protein consisting of the GAL4 -AD and the connection of a possible binding partner for the bait protein ( prey protein prey protein). Both yeast two- hybrid plasmids replicate both in yeast and in E. coli autonomous ( shuttle vectors ).

A yeast strain which does not have a functional GAL4 gene and one or more reporter genes carries is transformed with both plasmids, to which a binding site is upstream of the GAL4 transcription factor. As reporter genes of this interaction function, which can produce either specific amino acids or bases (for example, histidine, uracil or adenine) or an optical detection enable (e.g., a color change catalyzed by the lacZ gene). When there is an interaction between the bait and prey, this results usually a functional reconstitution of the GAL4 transcription factor, resulting in expression of the reporter genes to the sequence. The latter can be detected by growth on appropriate selective media, such as on a histidine deficient medium grow in the sequence only yeasts in which bait and prey interact and so express the enzyme for histidine synthesis.

In a screening method possible interaction partners in a more empirical approach with a cDNA library as " Prey" are identified, or it may, at the so-called "single mating" with this system, specifically the interaction of certain proteins to be checked. The DNA sequences may be derived from a DNA purification or synthesis. There are variants of the Y2H in E. coli and mammalian cells.

Pros and Cons

The yeast two -hybrid system allows the study of protein- protein interactions in vivo, at least in similar circumstances, that is, in the milieu of a cell and occurring in eukaryotes, post-translational modifications such as glycosylation ( attachment of sugar chains), palmitoylation ( appending fatty acids) or by folding chaperones. Since yeast is also to use a relatively cheap and robust, many interacting partners, for example in screening approaches are checked.

A problem with the classical yeast two -hybrid system is that the interaction of the proteins under investigation must take place in the nucleus of yeast, since transcription can occur only there. However, it is possible that proteins fold differently in this environment than in the area of the cell in which they usually occur. The modifications of the yeast are partially different from those of other eukaryotic organisms. This change affects folding structure and surface properties of the proteins, which can lead to incorrect test results:

• Proteins that would not normally interact with each other to do this through the modified surface ( false positive)
• Proteins actually interact with each other, it can not by altered folding in the cell nucleus ( false negative)

Furthermore, it is possible that two proteins indeed interact in the Y2H experiment, but in the cell cycle, do not occur in the organelle or cell type at a time and therefore may not be actual interaction partners.

For these reasons, the interpretation of Y2H results with great caution must be made. Positive interactions should always be verified with other techniques from molecular biology such as immunoprecipitation or FRET. The Y2H experiment is still true information about the possible interaction of two proteins, but no information about how this interaction takes place. These detailed studies of the structure of the proteins involved are necessary.

The classic, described here yeast two -hybrid system has additional restrictions. For example, certain proteins can not be transported into the nucleus, such as membrane proteins. Also, protein complexes, involving more than two proteins are involved, can not directly examine with classical Y2H experiments. To address these problems, modified yeast two- hybrid systems have now been developed, in which only additional steps allow the reconstitution of the transcription factor. Also, there are now systems that are based on other principles, such as the split-ubiquitin system, which is particularly interesting for interaction studies of membrane proteins.