YEAST TWO-HYBRID

SYSTEM

Subham Kumar Tripathy

1311051
SBS,NISER
Yeast two-hybrid system is a screening technique
used to discover protein-protein interactions by
testing for physical interaction (binding) between
two proteins (hybrid).

Modular nature of Transcriptional activator proteins

play a major role .
AD
Transcription Factor
DBD
 AD

prey
transcription
factor
bait

DBD
Yeast Two-Hybrid System

bait prey
AD

DBD RNA polymerase

selectable marker
PROMOTER
Reporter Gene
Link between DBD & AD is
important

No recruitment of
RNA pol

Target sequence
binding doesnt
occur

Gene expression
occurs
Gal4 Gal4
DBD AD

prey
bait
 Co-Transformation of both the the plasmids

Reporter gene express Reporter

gene doesnt express

Proteins interact Proteins

dont interact
 Advantages in yeast:
Number of nutritional genes can be selected for
deficient hosts e.g. ura, trp,ade,lacZ
Large number of reporters are available for
yeast.
Limitations of Yeast Two-Hybrid
System:
High number of false positive identification.
Weak interactions between proteins.
Transmembrane protein interaction .
Multiple Reporter used in same cell to reduce the
false positive:
Multiple baits are used to reduce false
positive:
 Membrane protein interaction:
SOS recruitment system where Protein-protein
interaction occurs at membrane.
Plasmid 1 hSos fused with Targetprotein (bait)
Plasmid 2 prey protein fused with membrane
localizing sequence

cdc25 mutant yeast is taken.

hSos activates RAS which bypasses cdc25 pathway.
 One hybrid system:
Single fusion protein.
For protein-DNA interaction.
Constant- AD
Variable- DBD
Three hybrid system:
PROTEIN CRYSTALLIZATION
CRYSTALLIZATION
It is the process of obtaining crystals.

CRYSTALS
Orderly 3D arrangement of molecules in repeating
array held together by non-covalent bonds.

WHY CRYSTALS?
There is no detector which detect the diffraction
data of single molecule.
It is better to see more number of molecules than
a single molecule.
For crystallization gradual growth of
crytal form undersaturated zone to
nucleation zone is essential.
 METHODS FOR PROTEIN
CRYSTALLIZATION
Hanging drop :
Sitting Drop method
Other Methods
Microdialysis
Microbatch