The Split-HybridTM System
As Developed by Bio101 Inc.1
What is the Split-HybridTM System?
The Split-Hybrid System, developed by Bio 101 Inc.1, is a method developed to test for mutations, proteins, peptides and drugs which cause disruptions of protein-protein interactions in vivo within the yeast Saccharomyces cerevisiae1. The Split-Hybrid system is developed beyond previous protein interaction assays because it incorporates a positive selection method2. This positive selection mechanism allows for positive results where yeast colony growth occurs only in the presence of disruptions of proetin-protein interactions. This system is intended to improve the efficiency of three types of screening:
1) Screening for substances which cause a failure in protein
to protein interaction.
2) Mutagenesis Screening (Mutations which can result in growth on an appropriate media3.)
3) Screening libraries to isolate disruptive peptides
How does the Split-HybridTM System work?
of proteins with other proteins can be manipulated or disrupted by several
factors including mutations, proteins, peptides, and drugs. This
system is designed to detect errors in the protein interaction of specific
proteins of interest. This system expresses the proteins of interest
as fusion proteins. As described by the Split-Hybrid System webpage1,4,
the fusion protein of interest, X, binds to the pLEXA DNA construct, and
the fusion protein of interest, Y, binds to the transcription activator
VP16 pSHM.1-LacZ (Figure 1). The two constructs, pLEXA and VP16 pSHM.1-LacZ
can be seen in figure 2. The X and Y fusion complexes then bind to
the LexA operator which allows the transcription of the Tetracycline repressor
protein (TetR). If there is a mutation causing a defect in the fusion
of the X and Y complexes, then the LexA operator will not be induced to
transcribe the TetR gene. However, if the TetR is produced, it will
bind to the Tetracycline Operator and prevent the transcription of the
HIS3 reporter gene1,4. Without the ability to transcribe
the HIS3 reporter gene, the cell can not produce the amino acid histidine.
Thus, such cells could not grow on plates lacking histidine. This is demonstrated
in the "Interaction" section of Figure 1. However, in the presence
of interference factors for protein-protein interactions, the TetR repressor
would not be produced, and the HIS3 histidine gene would not be shut down.
This is demonstrated in the "Disruption" section of Figure 1. As
a result, only in the malfunction of protein-protein interaction will cells
be able to grow on plates lacking histidine. Thus, the Split-Hybrid
System is a positive expression system.
The Split-Hybrid System at work:
In Shih et al2, the split-hybrid system was used to find a phosphorylation mutation at Ser-133 involving the interaction of CREB and its coactivator binding protein (CBP). This paper compares the standard two-hybrid assay with the improved split-hybrid assay. The split-hybrid assay allowed growth of cells on media with histidine where as the two-hybrid assay did not allow growth of cells2. This demonstrates the positive selection improvement of the split-hybrid system.
New methods for detecting protein to
protein interactions in yeast originated from the paper "A Novel
Genetic System to Detect Protein-Protein Interactions" by Stanley Fields
and Ok-kyu Song in 19893. Fields and Ok-kyu used fusion hybrid
proteins of the GAL4 protein in yeast to detect protein interactions3.
Figure 1. The interaction of fusion proteins X and Y causes an inability of the cell to produce histidine, whereas the lack of interaction between X and Y leads to the production of histidine. This image was obtained with permission from the authors of Bio101 Inc. <http://www.bio101.com/dna-kits/split-hybrid.html>
1. 1999. Split-HybridTM Yeast Kit. <http://www.bio101.com/dna-kits/split-hybrid.html>
2000 Feb 16.
2. Shih H, Goldman P, DeMaggio A, Hollenberg S, Goodman R, Hoekstra M. A positive genetic
selection for disrupting protein-protein interactions: Identification of CREB mutations that prevent
association with the coactivator CBP. Proc. Natl. Acad. Sci. USA 1996 Nov. (93): 13896-13901.
3. Fields S, Ok-kyu S,. A novel genetic system to detect protein-protein interactions. Nature 1989
4.1999. Split-HybridTM System. <http://www.bio101.com/newsletter/function4/8.html> Accessed
2000 Feb 20.
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