Two-hybrid screening

# Overview
Two-hybrid screening or 2-hybrid screening is a molecular biology technique used to discover protein-protein interactions and protein-DNA interactions by testing for physical interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively.
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 the 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.

# History
Pioneered by Fields and Song in 1989, the technique was originally designed to detect protein-protein interactions using the GAL4 transcriptional activator of the yeast Saccharomyces cerevisiae. The GAL4 protein activated transcription of a protein involved in galactose utilization which formed the basis of selection. Since then, the same principle has been adapted to describe many alternative methods including some that detect protein-DNA interactions, DNA-DNA interactions and use Escherichia coli instead of yeast.

# Basic premise
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 often 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.
Plasmids are engineered to produce a protein product in which the DNA-binding domain (BD) fragment is fused onto a protein while another plasmid is engineered to produce a protein product in which the activation domain (AD) fragment is fused onto another protein. The protein fused to the BD may be referred to as the bait protein and is typically a known protein that the investigator is using to identify new binding partners. The protein fused to the AD may be referred to as the prey protein and can be either a single known protein or a library of known or unknown proteins. In this context, a library may consist of a collection of protein-encoding sequences that represent all the proteins expressed in a particular organism or tissue or may be generated by synthesising random DNA sequences. Regardless of the source, they are subsequently incorporated into the protein-encoding sequence of a plasmid which are then transfected into the cells chosen for the screening method. This technique, when using a library, assumes that each cell is transfected with no more than a single plasmid and that therefore, each cell ultimately expresses no more than a single member from the (or each, if two libraries are selected against one another) protein library.
If the bait and prey proteins interact (i.e. bind), then the AD and BD of the transcription factor are indirectly connected, bringing the AD in proximity to the transcription start site and transcription of reporter gene(s) can occur. If the two proteins do not interact, there is no transcription of the reporter gene. In this way, a successful interaction between the fused protein is linked to a change in the cell phenotype.
The challenge of separating those cells which express proteins which happen to interact with their counterpart fusion proteins, from those which do not, is addressed in the following section, 'Reporter and selection genes'.

# Reporter and selection genes
In order to link the interaction to a change in observable phenotype, a reporter gene is provided with the upstream activation sequence (UAS) to which the binding domain binds, resulting in gene expression in successful cases of interaction. Since its inception in 1989, the technique has been combined with a number of different reporter genes which can allow selection through a simple colour change or through automatic death of cells in which the interaction does or does not take place.
The lacZ reporter gene will allow the highlighting of cells in which the UAS-BD-AD interaction is taking place. -galactosidase, the gene product of lacZ produces a blue colouration through the metabolism of X-gal (5-bromo-4-chloro-3-indolyl--D-galactoside) which allows the experimenter to manually choose the individuals which host proteins displaying the required  interaction.
Manual differentiation of cells according to colour may be acceptable for small numbers of cells, as when investigating a small number of proteins, but when a large library of proteins must be screened, some automation is necessary. This automation is provided by a number of genes and gene systems which either cause the death of cells that aren't hosting interactions or vice versa, leaving only the cells expressing the proteins of interest.
For more autonomy, alternative reporter genes exist that enable automatic deletion of individuals that fail to express the reporter or vice versa.
Some projects utilise a dual reporter system in which two reporter genes are used in order to help substantiate the interaction. When developing their HIS3 selection system, Joung et al. (2000) included an aadA gene, downstream of the HIS3 gene such that it may be expressed at the same time as the HIS3 gene. The aadA gene confers spectinomycin resistance and although insufficient as a selection gene in itself, was used to maintain selection pressure and provide greater stringency.

## Positive selection
In positive selection, the individuals that host a successful interaction are able to live on the provided media, while those failing to host this interaction die. This can be achieved by combining a media that is lacking an essential nutrient with a strain of an organism which is dependent on expression of the reporter gene in order to produce this nutrient. Examples include the HIS3 gene, encoding a protein required for histidine synthesis, the LEU2 gene, encoding a protein required for leucine synthesis and the URA3 gene, encoding a protein required for uracil synthesis.
The positive selection method is used in investigations in which the investigators are aiming to discover proteins which interact. Applications include the discovery of homologues in other species and the discovery of members of a protein family (containing a particular domain and therefore binding to a compatible domain). This HIS3 gene for example can be used with E. coli cells bearing a deletion in their homologous hisB gene (hisB cells).

## Counter-selection
Alternatively, reporter genes conferring sensitivity to an agent supplied in the media, will kill cells expressing the reporter, thereby removing cells that host an interaction. Examples of genes used in the counter selection method include CYH2 (confers sensitivity to cycloheximide), CAN1 and URA3. Alternatively, using repressor domains or other negative regulators in place of activation domains, counter-selection may be achieved using the same reporter genes used in positive selection methods. One such negative regulator is the yeast GAL80 domain which binds and inactivates the transcriptional activator region of GAL4.
Such counter-selection methods may be used in investigations aiming to discover a mutation, chemical or protein that will interfere with the interaction.

# Fixed domains
In any study, some of the protein domains, those under investigation, will be varied according to the goals of the study whereas other domains, those that are not themselves being investigated, will be kept constant. For example in a two-hybrid study to select DNA-binding domains, the DNA-binding domain, BD, will be varied whilst the two interacting proteins, the bait and prey, will need to be kept constant in order to maintain a strong binding between the BD and AD. There are a number of domains from which to choose the BD, bait and prey and AD, if these are to remain constant. In protein-protein interaction investigations, the BD may be chosen from any of many strong DNA-binding domains such as Zif268. A frequent choice of bait and prey domains are residues 263-352 of yeast Gal11P with a N342V mutation and residues 58-97 of yeast Gal4, respectively. These domains can be used in both yeast- and bacterial-based selection techniques and are known to bind together strongly.
The AD chosen must be able to activate transcription of the reporter gene, using the cell's own transcription machinery. Thus, the variety of ADs available for use in yeast-based techniques may not be suited to use in their bacterial-based analogues. The herpes simplex virus-derived AD, VP16 and yeast Gal4 AD have been used with success in yeast whilst a portion of the -subunit of E. coli RNA polymerase has been utilised in E. coli-based methods.
Whilst powerfully-activating domains may allow greater sensitivity towards weaker interactions, conversely, a weaker AD may provide greater stringency.

# Construction of expression plasmids
There are a number of engineered genetic sequences which must be incorporated into the host cell in order to perform a two-hybrid analysis or one of its derivative techniques. The considerations and methods used in the construction and delivery of these sequences differ according to the needs of the assay and the organism chosen as the experimental background.
There are two broad categories of hybrid library: random libraries and cDNA-based libraries. A cDNA library is constituted by the cDNA produced through reverse transcription of mRNA collected from specific cells of types of cell. This library can be ligated into a construct so that it is attached to the BD or AD being used in the assay. A random library uses lengths of DNA of random sequence in place of these cDNA sections. A number of methods exist for the production of these random sequences, including cassette mutagenesis. Regardless of the source of the DNA library, it is ligated into the appropriate place in the relevant plasmid/phagemid using the appropriate restriction endonucleases.

## E. coli-specific considerations
By placing the hybrid proteins under the control of IPTG-inducible lac promoters, they are expressed only on media supplemented with IPTG. Further, by including different antibiotic resistance genes in each genetic construct, the growth of non-transformed cells is easily prevented through culture on media containing the corresponding antibiotics. This is particularly important for counter selection methods in which a lack of interaction is needed for cell survival.
The reporter gene may be inserted into the E. coli genome by first inserting it into an episome, a type of plasmid with the ability to incorporate itself into the bacterial cell genome with a copy number of approximately one per cell.
The hybrid expression phagemids can be electroporated into E. coli XL-1 Blue cells which after amplification and infection with VCS-M13 helper phage, will yield a stock of library phage. These phage will each contain one single-stranded member of the phagemid library.

# Recovery of protein information
Once the selection has been performed, the primary structure of the proteins which display the appropriate characteristics must be determined. This is achieved by retrieval of the protein-encoding sequences (as originally inserted) from the cells showing the appropriate phenotype.

## E. coli
The phagemid used to transform E. coli cells may be "rescued" from the selected cells by infecting them with VCS-M13 helper phage. The resulting phage particles that are produced contain the single-stranded phagemids and are used to infect XL-1 Blue cells. The double-stranded phagemids are subsequently collected from these XL-1 Blue cells, essentially reversing the process used to produce the original library phage. Finally, the DNA sequences are determined through dideoxy sequencing.

# Controlling sensitivity
The Escherichia coli-derived Tet-R repressor can be used in line with a conventional reporter gene and can be controlled by tetracycline or doxicycline (Tet-R inhibitors). Thus the expression of Tet-R is controlled by the standard two-hybrid system but the Tet-R in turn controls (represses) the expression of a previously mentioned reporter such as HIS3, through its Tet-R promoter. Tetracycline or its derivatives can then be used to regulate the sensitivity of a system utilising Tet-R.
Sensitivity may also be controlled by varying the dependency of the cells on their reporter genes. For example, this  effected by altering the concentration of histidine in the growth medium for his3-dependent cells and altering the concentration of streptomycin for aadA dependent cells. Selection-gene-dependency may also be controlled by applying an inhibitor of the selection gene at a suitable concentration. 3-Amino-1,2,4-triazole (3-AT) for example, is a competitive inhibitor of the HIS3-gene product and may be used to titrate the minimum level of HIS3 expression required for growth on histidine-deficient media.
Sensitivity may also be modulated by varying the number of operator sequences in the reporter DNA.

# Non-fusion proteins
A third, non-fusion protein may be co-expressed with the two fusion proteins. Depending on the investigation, the third protein may modify one of the fusion proteins or mediate or interfere with their interaction.
Coexpression of the third protein may be necessary for modification or activation of one or both of the fusion proteins. For example S. cerevisiae possesses no endogenous tyrosine kinase. If an investigation involves a protein that requires tyrosine phosphorylation, the kinase must be supplied in the form of a tyrosine kinase gene.
The non-fusion protein may mediate the interaction by binding both fusion proteins simultaneously, as in the case of ligand-dependent receptor dimerisation.
For a protein with an interacting partner, its functional homology to other proteins may be assessed by supplying the third protein in non-fusion form which then may or may not compete with the fusion-protein for its binding partner. Binding between the third protein and the other fusion protein will interrupt the formation of the reporter expression activation complex and thus reduce reporter expression, leading to the distinguishing change in phenotype.

# One-hybrid, three-hybrid and one-two-hybrid variants

## One-hybrid
The one-hybrid variation of this technique is designed to investigate protein-DNA interactions and uses a single fusion protein in which the AD is linked directly to the binding domain. The binding domain in this case however is not necessarily of fixed sequence as in two-hybrid protein-protein analysis but may be constituted by a library. This library can be selected against the desired target sequence which is inserted in the promoter region of the reporter gene construct. In a positive-selection system, a binding domain which successfully binds the UAS and allows transcription will thus be selected.
Note that selection of DNA-binding domains isn't necessarily performed using a one-hybrid system, but may also be performed using a two-hybrid system in which the binding domain is varied and the bait and prey proteins are kept constant.

## Three-hybrid
RNA-protein interactions have been investigated through a three-hybrid variation of the two-hybrid technique. In this case, a hybrid RNA molecule serves to adjoin together the two protein fusion domains which aren't intended to interact with each other but rather the intermediary RNA molecule (through their RNA-binding domains). Techniques involving non-fusion proteins that perform a similar function, as described in the 'non-fusion proteins' section above, may also be referred to as three-hybrid methods.

## One-two-hybrid
Simultaneous use of the one- and two-hybrid methods (that is, simultaneous protein-protein and protein-DNA interaction) is known as a one-two-hybrid approach and expected to increase the stringency of the screen.

# Host organism
Although theoretically, any living cell might be used as the background to a two-hybrid analysis, there are practical considerations that dictate which will be chosen. The chosen cell line should be relatively cheap and easy to culture and sufficiently robust to withstand application of the investigative methods and reagents.

## Yeast
S. cerevisiae was the model organism used during the two-hybrid technique's inception. It has several characteristics that make it a robust organism to host the interaction, including the ability to form tertiary protein structures, neutral internal pH, enhanced ability to form disulfide bonds and reduced-state glutathione among other cytosolic buffer factors, to maintain a hospitable internal environment. The yeast model can be manipulated through non-molecular techniques and its complete gene sequence is known. Yeast systems are tolerant of diverse culture conditions and harsh chemicals which could not be applied to mammalian tissue cultures.
Proteins from as small as eight to as large as 750 amino acids have been studied using yeast.

## E. coli
E. coli-based methods have several characteristics which may make them preferable to yeast-based homologues. The higher transformation efficiency and faster rate of growth lends E. coli to the use of larger libraries (in excess of 108). A low false positive rate of approximately 3x108, the absence of requirement for a nuclear localisation signal to be included in the protein sequence and the ability to study proteins that would be toxic to yeast may also be major factors to consider when choosing an experimental background organism.
It may be of note that the methylation activity of certain E. coli DNA methyltransferase proteins may interfere with some DNA-binding protein selections. If this is anticipated, the use of an E. coli strain that is defective for a particular methyltransferase may be an obvious solution.

# Applications

## Determination of sequences crucial for interaction
By changing specific amino acids by mutating the corresponding DNA base-pairs in the plasmids used, the importance of those amino acid residues in maintaining the interaction, can be determined.
After using bacterial cell-based method to select DNA-binding proteins, it is necessary to check the specificity of these domains as there is a limit to the extent to which the bacterial cell genome can act as a sink for domains with an affinity for other sequences (or indeed, a general affinity for DNA).

## Drug and poison discovery
Protein-protein signalling interactions pose suitable therapeutic targets due to their specificity and pervasiveness. The random drug discovery approach utilises compound banks that comprise random chemical structures and demands a high-throughput method in order to test these structures in their intended target.
The cell chosen for the investigation can be specifically engineered to mirror the molecular aspect that the investigator intends to study and then used to identify new human or animal therapeutics or anti-pest agents.

## Determination of protein function
By determination of the interaction partners of unknown proteins, the possible functions of these new proteins may be inferred. This can be done using a single known protein against a library of unknown proteins or conversely, by selecting from a library of known proteins using a single protein of unknown function.

## Zinc finger protein selection
To select zinc finger proteins (ZFPs) for protein engineering, methods adapted from the two-hybrid screening technique have been used with success. A ZFP is itself a DNA-binding protein used in the construction of custom DNA-binding domains that bind to a desired DNA sequence.
By using a selection gene with the desired target sequence included in the UAS and randomising the relevant amino acid sequences to produce a ZFP library, cells that host a DNA-ZFP interaction with the required characteristics can be selected. Each ZFP typically recognises only 3-4 base pairs, so to prevent recognition of sites outside the UAS, the randomised ZFP is engineered into a 'scaffold' consisting of another two ZFPs of constant sequence. The UAS is thus designed to include the target sequence of the constant scaffold in addition to the sequence for which a ZFP is being selected.
A number of other DNA-binding domains may also be investigated using this system.

# 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 criticism applied to the yeast two-hybrid screen of protein-protein interactions is the possibility of a 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%. 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.