The document discusses several techniques for studying protein-protein interactions, including the yeast two-hybrid system. It describes the basic procedure of the yeast two-hybrid system and some advantages, such as its ability to detect weak and transient interactions in vivo. The document also summarizes several modifications to the original yeast two-hybrid system, including the use of three proteins, dual bait systems, and recruitment systems to address some of the limitations and disadvantages of the classical yeast two-hybrid approach.

2. • THE LARGE SCALE STUDY OF PROTEIN ITS STRUCTURE AND
FUNCTION AND PROTEIN-PROTEIN INTERACTION.

3. In the late 1990s the protein function analyses is mainly focusing on the single protein.
But because the majority protein perform their function properly with interact with other protein.
Now the understanding the function of protein is should be studied to their interacted proteins.
With the development to the field of protein help to understand how the protein interact with each
other and also identifying the biological network is vital to understand how protein function within
the cell.

4.  Biochemical & Biophysical approaches
 Affinity Chromatography
 Fluorescence resonance Energy transfer (FRET)
 Surface Plasma Resonance
 Atomic Force Microscopy (AFM)
 X-ray Diffraction
 Molecular genetic approaches
 Yeast Two Hybrid System

5. • The Yeast two Hybrid system have clear advantage over biochemical and or genetic
method,
• It is in vivo technique uses yeast cell in the living test tube.
• It have a greater resemble to the higher eukaryotic organism than a system based on
bacterial host.
• The biochemical approaches required higher quality of purified protein while yeast
two hybrid system minimal requirement for screening ,such as the cdna of the gene
of interest which needed.

6. CONTINUE…
• Weak and transient interaction is very important. These interaction can also be
detected through Hybrid system.
• The yeast hybrid system is also useful for the known interaction analysis which can
be achieved by modification of residues or observing the effect of binding site.
Two hybrid screen sometime called functional screens because if a protein is screened
has known function in a well define pathway they provide a hint to the function of
current interaction.

7. • Yeasts are eukaryotic, single-celled microorganism.
• Saccharomyces cerevisiae is most studied eukaryotic model organism like E-coli as
model for prokaryotic,
• Saccharomyces cerevisiae cell is round to oval, 5-10 micrometer in diameter.
• It reproduce by a division process known as budding.
• Saccharomyces cerevisiae was first eukaryotic genome was completely sequenced in
April 1996.
• The genome is composed of about 13,000,000 and about 6,275 genes.

15. Summary

16. Applications of classical y2h
system

19. Modifications of theYeastTwo-Hybrid system
ThehSosrecruitment system
Three protein system
Thedual-bait system

20. ThehSos recruitmentsystem
•Plasmid encoding Xfused in-frame to (RasGEF)hSos
•Plasmid encoding Yfused in-frame with av-Srcmyristylationsignal

21. Procedure
Twoplasmids are constructed, one encoding protein Xfused in-frame to the human Rasguanyl
nucleotide exchangefactor (RasGEF)hSos,the secondencoding protein Yfused in-frame with av-
Srcmyristylationsignal.
Theplasmids are co-transformed into atemperature-sensitive yeast mutant containing alesion in
the Cdc25 genewhichencodes ayeast Ras GEF.
Theprotein Yfusion is targeted to the yeast plasma-membrane.Interaction between proteins Xand Y
recruits hSosto the yeast plasma membrane where it complements the cdc25 mutation by
activating the Ras signalling cascade.
Interactionsare detected through growth of yeast cells at the restrictive temperature (37◦C).

22. Applications
Cytoplasm-based yeast two-hybrid system
Donot rely on atranscriptionalreadout
Transcriptional repressors
Auto-activation of reporter genesby baitconstructs
Theproblem of certain proteins not localizing to the yeast cell nucleus

23. Threeproteinsystem

24. Procedure
Thissystemis basedon the classical yeast two-hybrid system.ProteinsXand Yare expressedin-
frame with atranscription factor DNA-binding domain and transcription activation domain,
respectively.
Athird protein, Z,is expressedwith anuclear localization signal,without
any added domains, in the yeastnucleus.
Protein Ymayonly interact with Xin the presenceofZ.
(i) Adomain formed through the interaction between Xand Zmayprovide an interaction interface
for proteinY.
(ii) Alternatively, protein Zmay act asabridge between proteins XandY

25. Thedual-baitsystem
X1fused to DNA- binding
domainLexA
X2fused to DNA-
binding domainλcI

26. Procedure
Twotest proteins (X1and X2)are fused to two differentDNA-binding domains (LexAand λcI,
respectively).
The two fusion constructs are co-expressed in the same yeast cell and tested for interaction with
proteins fused to the B42transcriptionactivation domain.
Interaction with X1induces expression of LexA-dependent reportergenes (lacZ andLEU2).
Interaction with X2induces expression of λcI-dependent reportergenes (LYS2 and gusA)

27. Applications
Twotest proteins canbe analyzed for protein-protein interactionpartners in asingle library
screening
Totest the specificity of aprotein-protein interactionamongst
evolutionarily conservedproteins
Toidentify domains or residues required for interaction withone partner but notanother

28. Advantages
Immediate availability of the cloned gene of the interacting protein
Only asingle plasmid construction isrequired
Interactions are detected invivo
Weak,transient interactions canbedetected
Canaccumulate aweak signal over time
Protein purification notnecessary
No antibodies requries
Twohybrid systems - >touncover unanticipated
interactions

29. Disadvantages
Falsepositives are the largest problem with the yeast two-hybrid system.Canbe caused
by the ability of bait toinduce transcription without interaction with thebait
Possibleincorrect protein folding inyeast
gene encoding target protein must beavailable
failed to detect some knowinteractions
Elimination of FalsePositives
SequenceAnalysis
Retransformation of both strain with bait plasmid and strain without bait plasmid
Testfor interaction with an unrelated proteinasbait

30. Thank youTHE END