basic information about Yeast two hybrid

1. Presentation
on
Submitted by :
Desai Vruddhi K.
M.Sc.(Agri), 3rd sem.
Reg.No:04-AGRMA-01581-17
Dept. of GPB,
C.P.C.A, SDAU

2. proteomics
 Proteomics is the large-scale study of proteins, particularly their
structures and functions. Proteins are vital parts of living
organisms, as they are the main components of the physiological
metabolic pathways of cells

3. 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.

4. 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)

5. How does it work?
 Uses yeast as a model for eukaryotic protein
interactions
 A library is screened or a protein is
characterized using a bait construct
 Interactions are identified by the
transcription of reporter genes
 Positives are selected using differential
media

6. The Model
DNA-Binding
Domain
Bait Protein Prey Protein
Transcription
Activating
Region
Reporter Gene
DNA-Binding Site

7. Steps to Screen a Library
 Create the Bait Plasmid Construct from the
gene of interest and the DNA binding
domain of Gal4 or LexA or other suitable
domain
 Transform with the bait construct a yeast
strain lacking the promoter for the reporter
genes and select for transformed yeast
 Transform the yeast again with the library
plasmids
 Select for interaction

8. Reporter Genes
 LacZ reporter - Blue/White Screening
 HIS3 reporter - Screen on His+ media
(usually need to add 3AT to increase
selectivity)
 LEU2 reporter - Screen on Leu+ media
 ADE2 reporter - Screen on Ade+ media
 URA3 reporter - Screen on Ura+ media

9. The classical yeast two-hybrid system
The early yeast two-
hybrid systems were
based on the finding
that many eukaryotic
factors have
separable bing DNA
transcription
activation domains
and transcription
activation domains.

10. •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.

12. 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.

16. Overall summary of yeast two
hybrid experiment
• Yeast two-hybrid
experiments yield
information on protein
protein interactions
• GAL4 Binding Domain
•GAL4 Activation Domain
•X and Y are two proteins of
interest
• If X & Y interact then
reporter gene is expressed

17. 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

18. 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.

19. Modifications of the Yeast Two-Hybrid system
Three protein system
The dual-bait system
The reverse two-hybrid
system

20. Three protein system

21. Procedure
This system is based on the classical yeast two-hybrid system.
Proteins X and Y are expressed in-frame with a transcription
factor DNA-binding domain and transcription activation
domain, respectively.
A third protein, Z, is expressed with a nuclear localization
signal, without any added domains, in the yeast nucleus.
Protein Y may only interact with X in the presence of Z.
 (i) A domain formed through the interaction between X
and Z may provide an interaction interface for protein Y.
 (ii) Alternatively, protein Z may act as a bridge between
proteins X and Y

22. The dual-bait system
 X1 fused to
DNA- binding
domain LexA
X2 fused to
DNA-binding
domain λcI

23. Procedure
 Two test proteins (X1 and X2) are fused to two
different DNA-binding domains (LexA and λcI,
respectively).
The two fusion constructs are co-expressed in
the same yeast cell and tested for interaction
with proteins fused to the B42 transcription
activation domain.
Interaction with X1 induces expression of LexA-
dependent reporter genes (lacZ and LEU2).
Interaction with X2 induces expression of λcI-
dependent reporter genes (LYS2 and gusA)

24. Applications
Two test proteins can be analyzed for protein-protein
interaction partners in a single library screening
To test the specificity of a protein-protein interaction amongst
evolutionarily conserved proteins
To identify domains or residues required for interaction with
one partner but not another

25. The reverse two-hybrid system

26. Procedure
This system is based on the classical two-hybrid
system except that expression of the reporter
gene is toxic to the yeast cells under certain
conditions.
In this example, the reporter gene URA3 allows
for selection of protein- protein interactions
between X and Y on media minus uracil and
counter- selection of disrupted protein-protein
interaction between X and Y on media
containing 5FOA.

27. Applications
This system can be used to identify residues required
for protein-protein interaction by making use of a
mutagenised copy of the cDNA encoding one of the
proteins.
cDNAs encoding proteins no longer able to interact can
be sequenced to reveal amino acids essential for
interaction.

28. Examples of Uses of the Yeast Two-Hybrid System
 Identification of caspase substrates
 Interaction of Calmodulin and L-Isoaspartyl
Methyltransferase
 Genetic characterization of mutations in E2F1
 Peptide hormone-receptor interactions
 Pha-4 interactions in C. elegans

29. advantage
 The two-hybrid system is popular due to its flexibility and rapid
isolation of interacting proteins.
 As the technique is used to identify protein interactions in a living
yeast cell, it offers a number of advantages, including protein
purification and antibody development at low cost, as well as a less
time consuming method of detecting of novel interacting proteins,
compared with conventional biochemical and genetic methods.
 It is an in vivo technique involving yeast as a host test tube. Yeast
cells represent a higher eukaryotic form, which exhibit the reality
closer than in vitro approaches or bacterial expression techniques.
 The technique is also represented as functional screens.
 The most significant features of the two-hybrid system are the two-
pronged technique of identifying an interacting protein and cloning
of the gene.

30. disadvantages
 It cannot provide a solution for all of the protein–
protein issues.
 One of the most significant drawbacks of this
technique is determining whether the specific
protein of interest can initiate transcription. The
approach can be successful only when the protein
activates the transcription on its own.
 One of the major disadvantages is the use
of Saccharomyces cerevisiae as the host cell; the
protein of interest must be able to fold correctly
with stability within the yeast cell.
 Another downside is that some protein interactions
depend on post-translational modifications that
may not occur or occur unsuitably in yeast.