YEAST TWO HYBRID METHODVipul choudhary | Dr. Indrakant K. Singh( سنگھ ( اندراکانت

Why we are studying protein-protein interactions?To fully understand their function

Need To Study PPI

The majority of proteins interact with other proteins for proper function, they should be studied in the context of their interacting partners to fully understand their function.

With the development of the field of proteomics, understanding how proteins interact with each other and identifying biological networks is vital to understanding how proteins function within the cell

Why Yeast Two Hybrid Method ?

It bears a greater resemblance to higher eukaryotic systems than a system based on a bacterial host.

With regards to classical biochemical approaches, which can require high quantities of purified proteins or good quality anti-bodies, the two hybrid system has minimal requirements to initiate screening, since only the cDNA of the gene of interest is needed.

The classical yeast two-hybrid system

The early yeast two-hybrid systems were based on the finding that many eukaryotic transcription factors have separable DNA-binding and transcription activationdomains.

•The protein of interest, the “bait”, is fused to a DNA-binding domain.

•Proteins that bind to bait, the “fish” or “prey”, are fused to a transcription activation domain.

•Proteins that do not bind to the bait will not activate the transcription of the reporter gene (HIS in this case)

•Any protein that binds to the bait will activate the transcription of the reporter gene

(a) No binding

(b) Binding between protein and bait

The first step is to construct a bait plasmid and a library.

Each type of plasmid contains a selectable marker such as an essential amino acid.

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Major applications of classical system

• Used to determine whether two known proteins interact with one another

• Used to identify unknown proteins, encoded by a cDNA library, that interact with a protein of interest

Powerful tool for investigating the network of interactions that form between proteins involved in particular biological processes

Selection of host strain

• The yeast strains used for two-hybrid experiments carry mutations in a number of genes required for amino acid biosynthesis, such as TRP1, LEU2, HIS3 and URA3.

• If these amino acids are omitted from the growth medium the yeast strain will fail to grow.

• Many of the two-hybrid plasmids carry genes that complement these mutations and allow for selection of the transformant yeast.

What is the yeast two-hybrid system used for ? Identifies novel protein-protein interactions Can identify protein cascades Identifies mutations that affect protein-protein binding Can identify interfering proteins in known interactions (Reverse

Two-Hybrid System)

Advantages

Immediate availability of the cloned gene of the interacting protein

Only a single plasmid construction is required Interactions are detected in vivo Weak, transient interactions can be detected Can accumulate a weak signal over time Protein purification not necessary No antibodies requires

Two hybrid systems - > to uncover unanticipated

interactions

Disadvantages

False positives are the largest problem with the yeast two-hybrid system. Can be caused by the ability of bait to induce transcription without interaction with the bait

Possible incorrect protein folding in yeast gene encoding target protein must be available failed to detect some know interactions