Warning: This web page was produced as an assignment for an undergraduate course at Davidson College.

This Page, composed by John McKillop is a description of :

The Split-Hybrid^TM System
As Developed by Bio101 Inc.¹

What is the Split-Hybrid^TM 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-Hybrid^TM System work?

The interaction 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 webpage^1,4, 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 gene^1,4. 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.

Background Information:

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>

Figure 2. Image of the pLEXA and pSHM.1-LacZ constructs.
This image was obtained with permission from the authors of Bio101 Inc. <http://www.bio101.com/newsletter/function4/8.html>

References

1. 1999. Split-Hybrid^TM Yeast Kit. <http://www.bio101.com/dna-kits/split-hybrid.html> Accessed
    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
    (340): 245-246.
4.1999. Split-Hybrid^TM System. <http://www.bio101.com/newsletter/function4/8.html> Accessed
   2000 Feb 20.

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