Two-hybrid screening
From Wikipedia, the free encyclopedia
Two-hybrid screening is a molecular biology technique used to discover protein-protein interactions by testing for physical interactions (such as binding) between two proteins. One protein is termed the bait and the other is a prey or library.
The premise behind the test is the activation of downstream reporter gene(s) by the binding of a transcription factor onto an upstream activating sequence (UAS). For the purposes of two-hybrid screening, the transcription factor is split into two separate fragments, called Binding Domain (BD) and Activating Domain (AD). The BD is the domain responsible for binding to the UAS and the AD is the domain responsible for activation of transcription.
The key to the two-hybrid screen is that in most eukaryotic transcription factors, the activating and binding domains are modular and can function in close proximity to each other without direct binding. This means that even though the transcription factor is split into two fragments, it can still activate transcription when the two fragments are indirectly connected.
The most common screening approach is the yeast two-hybrid assay. This system utilizes a genetically engineered strain of yeast in which the biosynthesis of certain nutrients (usually amino acids or nucleic acids) is lacking. When grown on media that lacks these nutrients, the yeast fail to survive. This mutant yeast strain can be made to incorporate foreign DNA in the form of plasmids. In yeast two-hybrid screening, separate bait and prey plasmids are simultaneously introduced into the mutant yeast strain. Each plasmid contains separate genes allowing synthesis of certain nutrients. One common system utilizes bait plasmid that synthesizes the amino acid tryptophan, and the prey plasmid synthesizes the amino acid leucine. When transformed together into yeast, these plasmids allow the mutant yeast to grow on media lacking both tryptophan and leucine.
Bait plasmids are also engineered to produce a protein product in which the BD fragment is fused onto the bait protein. Prey plasmids are engineered to produce a protein product in which the AD fragment is fused onto the prey protein. Whereas the bait protein is typically a known protein that the investigator is using to identify new binding partners, the prey protein can be either a known protein or a random library protein. If the bait and prey proteins interact (i.e. bind), then the AD and BD of the transcription factor are indirectly connected and transcription of reporter gene(s) occurs. If the two proteins do not interact, there is no transcription of the reporter gene. Typically, reporter genes encode for enzymes that allow synthesis of other specific nutrients that the mutant yeast strain is otherwise unable to produce. In one system, reporter genes include histidine and adenine biosynthesis. Thus, if two proteins in a screen interact, yeast containing these proteins will grow on media lacking tryptophan (allowed by the bait plasmid), leucine (allowed by the prey plasmid), adenine and histidine (allowed by interaction between bait and prey proteins, driving reporter genes). A screen in which there is no interaction between bait and prey proteins would yield yeast that grow on media lacking tryptophan and leucine only (because without bait/prey interaction, the AD and BD do not form a functional transcription factor allowing reporter nutrient biosynthesis). Another reporter gene includes an enzyme system to produce blue color - interactions result in yeast colonies that can generate blue color under certain conditions.
With a certain bait protein, two hybrid screening can be "directed" to test for protein-protein interaction with a known protein inserted into prey plasmid. Alternatively, "library screening" involves pairing bait protein with millions of different prey plasmids that have been engineered to produce protein from a unique, randomly inserted DNA fragment. The DNA fragments in library prey plasmids are synthesized from messenger RNA from a specific organism or tissue. As such they represent a "library" of the proteins expressed in a given organism or tissue and allow investigators to search for new protein-protein interactions from amongst all the proteins expressed in the species or tissue of choice.
Related techniques include one-hybrid screening and three-hybrid screening. The former tests directly for interaction between the library protein and a DNA target and the latter includes a third protein that bridges the bait and library proteins.
A common transcription factor used for yeast two-hybrid screening is GAL4.
Strengths and weaknesses
Two-hybrid screens are now routinely performed in many labs. They can provide an important first hint for the identification of interaction partners. Moreover, the assay is scalable which makes it possible to screen for interactions among many proteins.
The main weakness of the screen is the high number of false positive (and false negative) identifications. The exact rate of false positive results is not known, but estimates are as high as 50 % (Deane et al., 2002). The reason for this high error rate lies in the principle of the screen: The assay investigates the interaction between (i) overexpressed (ii) fusion proteins in the (iii) yeast (iv) nucleus. Each of these points (i-iv) alone can give rise to false results. For example, overexpression can result in non-specific interactions. Moreover, a mammalian protein is sometimes not correctly modified in yeast (e.g. missing phosphorylation), which can also lead to false results. Finally, some proteins might specifically interact when they are co-expressed in the yeast, although in reality they are never present in the same cell at the same time. Due to the combined effects of all error sources the overall confidence of the yeast two-hybrid assay is rather low. This means that all interactions have to be confirmed by a high confidence assay like co-immunoprecipitation of the endogenous proteins. However, this is a difficult task, especially for large scale protein-protein interaction data.
[edit] See also
[edit] External links
- Detail on sister technique two-hybrid system
- Science Creative Quarterly's overview of the yeast two hybrid system
[edit] References
- Deane CM, Salwinski L, Xenarios I, Eisenberg D. (2002) Protein interactions: two methods for assessment of the reliability of high throughput observations. Mol Cell Proteomics. 1:349-56. PMID: 12118076
