The document discusses the yeast two-hybrid (Y2H) system, a molecular biology technique used to identify protein-protein interactions (PPIs) critical for understanding biological activities and diseases. The Y2H system allows for the detection of these interactions in vivo using reporter genes, providing advantages such as minimal requirements for screening and the ability to detect weak interactions. However, challenges like false positives and potential incorrect protein folding in yeast are also highlighted, along with the conclusion that this method aids in examining cellular processes and discovering pathways.

1. Experimental technique  Yeast two-hybrid system
to identify Protein-Protein interactions

2. Protein-Protein interactions
 Protein–protein interactions are the
physical contacts of high specificity,
established between two or more
protein molecules.
 Proteins control and mediate many
of the biological activities of cells by
these interactions.
 Information about PPIs improves
our understanding of diseases and
can provide the basis for new
therapeutic approaches.
 Aberrant PPIs are the basis of
multiple aggregation-related
diseases, such as Creutzfeldt–
Jakob, Alzheimer's disease, and
may lead to cancer.

3. Examples of protein-protein interactions
Signal Transduction: activity of cells is regulated
through this way in form of extracellular signals
Transport across cell membrane: a protein may be
carrying another protein
Cell metabolism: In many metabolic pathways
different proteins interact to perform a specific
function.
Muscles contraction: myosin filaments act as
molecular motors and by binding to actin enable
filament sliding.

4. What is
Yeast Two
Hybrid
system?
 Also known as Y2H or Two-hybrid screening
 molecular biology technique
 Two Hybrid system is for Isolating and Studying Protein-Protein
Interactions.
 This system uses a reporter gene for detecting the interaction of
pair of proteins in yeast cell nucleus.
 Y2H assay relies on the expression of a reporter gene (such as
lacZ or GFP), which is activated by the binding of a particular
transcription factor.

5. Why Yeast Two Hybrid system
 The yeast two hybrid system has a clear advantage
over classical biochemical or genetic methods
 It is an in vivo technique that uses the yeast cell as a
living test-tube.
 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.

6. How 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.
 Interactions are identified by the
transcription of reporter genes.
 Positives are selected using
differential media.

7. 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 activation

8.  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

9.  Proteins that do not bind to the
bait will not activate the
transcription of the reporter
gene
 Any protein that binds to the
bait will activate the
transcription of the reporter
gene

10.  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.

15. 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

16. 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
 Elimination of False Positives
 Sequence Analysis
 Retransformation of both strain with bait plasmid and strain without bait plasmid
 Test for interaction with an unrelated protein as bait

17. Conclusion:
 The predictive power of the interactome model allows us to examine
the organization and coordination of multiple complex cellular
processes and determine how they are organized into pathways.
 The interactome model can be used to predict poorly characterized
proteins into their functional context according to their interacting
partners within a module.
 One-to-many relationship can be used for pathway discovery.