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Dual-Reporter 2-Hybrid Assay:
Although the two-hybrid assay has been useful in discovering protein-protein interactions, a major disadvantage of the approach is the large number of false-positives that arise. Technical false positives activate the transcription of the HIS3 reporter gene but do not represent physical interaction of the two proteins (“bait” and “prey”). To address this issue, we have been working on incorporating a second reporter into the existing system. The second reporter relies on the re-association of the firefly luciferase protein, which has been split in two halves and fused to the bait and prey constructs. With a true bait and prey protein-protein interaction, the two halves of luciferase are brought into proximity and should reconstitute enzyme activity. Thus, true physical interactions should be both transcription positive and luminescence positive, whereas false positives would yield only the transcriptional signal.
Initial tests revealed that the two halves of luciferase were capable of self-associating even in the absence of interacting bait and prey proteins. Splitting of the luciferase protein also led to a large decrease in luminescence upon re-association of the two halves, compared to the intact protein. To address these issues, I performed random mutagenesis on the luciferase gene in order to identify mutations that could either boost luminescence or decrease self-association. Mutations from these two classes were then coupled and the resulting constructs are being tested on several known bait/prey protein pairs as well as on yeast prey proteins that are known false positives. Validation of this new system will require a two-hybrid screen of a library to be performed, yielding results that are enriched in true positives.
Array Screening:
We constructed an array of ~6000 yeast transformants, each designed to express one of the S. cerevisiae open reading frames as a fusion to the Gal4 activation domain (AD). Using robotics, we can carry out a genomewide two-hybrid screen. A yeast strain expressing Gal4 DNA-binding domain (DBD) fused to any protein of interest is mated to the transformants in the array. Diploid cells are selected, pinned onto media selective for the two-hybrid interaction (-histidine) and scored for growth . Only cells that express interacting proteins should grow in the selective plates. The identity of interacting proteins is revealed by the positions of His+ colonies in the array. The initial description of the array, along with a collaborative effort by CuraGen, Inc. to carry out high throughput two-hybrid screens of yeast proteins, is described in Uetz et al. (2000).
*The data set can be downloaded here*
To date we have screened over 1000 yeast and several non-yeast proteins against the array. We are continuing this work on a collaborative basis through the Yeast Resource Center (YRC). Researchers with a specific gene of interest can contact us via the YRC website to request a collaboration. The gene of interest needs to be cloned into an appropriate two-hybrid DBD vector that we can provide. The identification of putative protein-protein interactions should further guide investigation of function.
In addition, we are working towards the development of a ‘dual-reporter’ Y2H system that should allow us to eliminate technical false positives that do not arise from a true interaction between the bait and prey proteins. Unlike the current 2-hybrid reporter system, this second reporter is not based on transcriptional activation, but rather on the reconstitution of a protein whose function can be selected or screened for. N-terminal and C-terminal fragments of proteins such as GFP and luciferase have been previously shown to function in split-reporter systems. We are in the process of fusing these domains to a well-established yeast two-hybrid protein pair and are currently testing different combinations in order to identify the optimal conditions that will allow us to distinguish between true and false positives.
Other publications describing yeast protein interactions that include work from the laboratory are listed here.
How the two-hybrid system works
How to clone 6000 ORFs
Primers • Vectors: pOBD2 and pOAD • Yeast strains • Recombination Cloning Protocols
•Sara Cooper and Sven Nelson
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