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Genetic screening systems for protein interaction discoveryYeast as a tool to identify novel protein interactions
Bakers yeast
Bakers yeast, Saccharomyces cerevisiae, is a unicellular eukaryote which has been widely used as a model system to elucidate many biological processes, such as intracellular signalling, cell cycle control, DNA replication and repair or protein translation.
Its easy genetic manipulation by various means has also made it the choice organism for methods that detect novel protein-protein interactions by means of genetic selection.
Detecting protein-protein interactions in yeast
Protein-protein interactions are intrinsic to virtually every cellular process within a cell and the formation of most cellular structures result from complex interactions between many different proteins. Traditionally, the tools available to analyze protein-protein interactions in multicellular organisms have been restricted to biochemical approaches. However, biochemical approaches can be difficult to set up and time-consuming to carry out.
The identification of protein-protein interactions by means of genetic selection in yeast has several important advantages. - Researcher's bias is removed: there is no need to manipulate binding or washing conditions, since interactions are detected in vivo.
- Proteins under investigation do not have to be purified and there is no need for cumbersome detection steps after complex purification.
- Novel protein-protein interactions can be easily selected from a pool of potential interaction partners (e.g. a cDNA expression library).
- Genetic screening systems yield not only information on the interaction itself but also directly provide the cDNA encoding the novel interaction partner.
The yeast two-hybrid system
Genetic selection for protein-protein interactions in yeast was first demonstrated by Fields and Song (1989) using a method they termed the yeast two-hybrid system. Since its invention in 1989, it has rapidly become the choice method for identifying novel protein-protein interactions.
The split-ubiquitin system and its adaptations
A wide range of similar screening systems have followed, one of them being the split-ubiquitin assay (Johnsson and Varshavsky, 1994). The split-ubiquitin assay is the basis for other genetic methods, such as the DUALmembrane system and a the DUALhunter system.
The yeast two-hybrid system Our DUALhybrid technology is based on the yeast two-hybrid system originally developed by Fields and Song (1989). The yeast two-hybrid system takes advantage of the modular nature of eukaryotic transcription factors to achieve a selection for protein-protein interactions.
Applications - Identify novel interaction partners of soluble proteins, protein domains or fragments
- Map binding interfaces
- Identify ternary protein complexes
- Identify small molecules that modulate protein interactions
>> Tell me more about the yeast two-hybrid system |
The DUALmembrane system The DUALmembrane system is based on the split-ubiquitin asay. The DUALmembrane system removes the need for interacting proteins to be located inside the nucleus. For this reason, it can be used to isolate interacting proteins from most environments inthe cell.
Applications - Identify novel interaction partners of integral membrane proteins
- Map binding interfaces
- Identify ternary protein complexes
- Identify small molecules that modulate protein interactions
>> Tell me more about the DUALmembrane system |
The DUALhunter system The DUALhunter system is an adaptation of the DUALmembrane system. An otherwise soluble bait is attached to the membrane by means of a small membrane anchor and screened against a cDNA library to identify interacting proteins. Because the DUALhunter system detects a protein interaction outside of the nucleus, it is especially suited for proteins that are self-activating in the classical yeast two-hybrid system.
Applications - Identify novel interaction partners of nuclear proteins, acidic proteins and generally self-activating proteins
- Identify novel interaction partners of membrane-associated proteins
- Map binding interfaces
- Identify ternary protein complexes
- Identify small molecules that modulate protein interactions
>> Tell me more about the DUALhunter system |
The split-ubiquitin system The split-ubiquitin system was developed in 1994 by Nils Johnsson and Alexander Varshavsky at Caltech. It provides a new approach to study protein-protein interactions.
The split-ubiquitin system is based on ubiquitin (Ub), a small, highly conserved protein that is attached to lysine residues of proteins in order to tag them for proteasomal degradation. Ubiquitin-tagged proteins are recognized by ubiquitin-specific proteases (UBPs) that cleave after the C-terminal (Gly 76) residue of Ub and the first residue of the target protein. Johnsson and Varshavsky found that native ubiquitin can be split into an N-terminal (Nub) and a C-terminal (Cub) half. The two halves retain a basic affinity for each other and spontaneously reassemble to form quasi-native ubiquitin. Modifications in the Nub part lead to a weakening of this reconstitution and provide the basis for studying protein-protein interactions by means of ubiquitin reassembly. >> Tell me more about the split-ubiquitin system |
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