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Thursday, July 31, 2014

Yeast Two Hybrid System - For Protein - Protein Interaction Studies

Yeast Two Hybrid system uses a reporter gene to detect the interaction of pair of proteins inside the yeast cell nucleus. In the yeast Two Hybrid System, The interaction of target protein to the protein will bring together transcriptional activator, which then switches on the expression of reporter gene.

The Yeast Two Hybrid system makes use of modular nature of gene activator proteins. These proteins bind to the DNA and activate the transcription of the gene.

How the Yeast Two Hybrid System Works

Even though it looks complex, Yeast Two Hybrid System is relatively simple to use in labs to study protein-protein interaction. As the protein-protein interaction occurs inside the yeast cell nucleus, proteins from every part of the cell and from any organism can be studied using yeast two hybrid system.

This is how the Yeast Two Hybrid System works:

The DNA sequence that encodes target protein is fused with the DNA that encodes the DNA-binding domain of gene activator protein using recombinant DNA technology.

Two sets of proteins are used:

·         Bait and the Prey

Target protein is fused to a DNA-binding domain that localizes it to the regulatory region of a reporter gene as “bait.”

Specially designed protein in the cell nucleus (“prey”).

Bait can bind to the regulatory region of the reporter gene, which acts as a bait and can be used to fish out the protein that interact with the target protein inside yeast cell. The interaction of Bait and Prey will bring about the activation and expression / transcription of reporter gene. The reporter gene is the one which will help the yeast cell grow on selective medium.

Many potential binding partners can be prepared by ligating DNA encoding the activation domain of gene activator protein to a large mixture of DNA fragments from a cDNA library.

Cells that express this reporter are selected and grown, and the gene (or gene
fragment) encoding the prey protein is retrieved and identified through
nucleotide sequencing.
Through Two Hybrid System a protein linkage map has been generated for most of the 6,000 proteins in yeast and similar projects are underway to catalogue the protein interactions in C. elegans and Drosophila.

A similar technique called reverse two hybrid system, which can be used to detect mutation of chemical compounds that are able to disrupt specific protein interactions. In reverse two hybrid system the reporter gene can be replaced with a gene that kills the cells when the bait and prey proteins interact. Eliminating a particular molecular interaction can reveal something about the role of the participating proteins in the cell. In addition, compounds that selectively interrupt protein interactions can be medically useful: a drug that prevents a virus from binding to its receptor protein on human cells could help people to avoid infections.

Alberts, Molecular Biology of the Cell
Cooper, Molecular Biology of the Cell
The Yeast Two-hybrid System - Paul L. Bartel, Stanley Fields

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