Two-hybrid screening
Pioneered by Stanley Fields and Ok-Kyu 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 gene involved in galactose utilization, which formed the basis of selection.
This mutant yeast strain can be made to incorporate foreign DNA in the form of plasmids.
[3] Regardless of the source, they are subsequently incorporated into the protein-encoding sequence of a plasmid, which is then transfected into the cells chosen for the screening method.
There are a number of domains from which to choose the BD, bait and prey and AD, if these are to remain constant.
A number of engineered genetic sequences must be incorporated into the host cell to perform 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.
[2] By placing the hybrid proteins under the control of IPTG-inducible lac promoters, they are expressed only on media supplemented with IPTG.
This is particularly important for counter selection methods in which a lack of interaction is needed for cell survival.
[2] The double-stranded phagemids are subsequently collected from these XL-1 Blue cells, essentially reversing the process used to produce the original library phage.
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.
For instance, the KInase Substrate Sensor ("KISS"), is a mammalian two-hybrid approach has been designed to map intracellular PPIs.
Here, a bait protein is fused to a kinase-containing portion of TYK2 and a prey is coupled to a gp130 cytokine receptor fragment.
[15] 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.
[1] Although theoretically, any living cell might be used as the background to a two-hybrid analysis, there are practical considerations that dictate which is 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.
Depending on the system used agar plates or specific growth medium is used to grow the cells and allow selection for interaction.
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.
Due to this different codon usage it is difficult to use the model system S. cerevisiae as a Y2H to check for protein-protein interactions using C. albicans genes.
[23][24][25] Bacterial two hybrid methods (B2H or BTH) are usually carried out in E. coli and have some advantages over yeast-based systems.
For instance, the higher transformation efficiency and faster rate of growth lends E. coli to the use of larger libraries (in excess of 108).
[32] The sea hare A californica is a model organism in neurobiology to study among others the molecular mechanisms of long-term memory.
To study interactions, important in neurology, in a more native environment a two-hybrid system has been developed in A californica neurons.
[1] 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).
[2] Protein–protein signalling interactions pose suitable therapeutic targets due to their specificity and pervasiveness.
[36] 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.
[2] The reason for this high error rate lies in the characteristics of the screen: Each of these points alone can give rise to false results.
Due to the combined effects of all error sources yeast two-hybrid have to be interpreted with caution.
A . The Gal4 transcription factor gene produces a two-domain protein ( BD and AD ) essential for transcription of the reporter gene ( LacZ ).
B , C . Two fusion proteins are prepared: Gal4BD+Bait and Gal4AD+Prey . Neither of them are usually sufficient to initiate transcription (of the reporter gene) alone.
D . When both fusion proteins are produced and the Bait part of the first fusion protein interacts with the Prey part of the second, transcription of the reporter gene occurs.
