Transcript Detection of protein-protein interactions

Protein-protein interactions
Why study protein interactions?
• To infer function
• To understand regulatory networks
Approach
With given bait, discover target
Methods
• Two-hybrid system
• Phage display
Two-hybrid system 1.target plasmid
• Target plasmid directs transcription of the Nterminal domain of RNA polymerase α –subunit
• The target gene is fused in-frame to RNAPα at its
the 3' end.
• A promoter controls transcription
Two-hybrid system
-2. bait vector
•The bait vector encodes a repressor protein,
to which the ‘bait’ protein of interest is fused.
• The promoter of the repressor protein
makes expression of the bait fusion inducible
by IPTG.
Two-hybrid system – 3. reporter system
• The E. coli reporter strain carries an auxotrophy
(His3) and a resistance gene (Strepr) on an F'
episome.
• This strain is transformed with a recombinant
target and a bait plasmid.
• Expression of target and bait protein fusions are
induced by IPTG.
• If bait and target interact, RNApol initiates
transcription of His3 and Strepr genes.
• Screening 1st for His prototrophy, 2nd for
streptomycin resistance.
Two-hybrid system – final step
• Sequence the ‘positive’ target plasmid to
determine the protein that interacts with a
given bait
Phage display
• Cloning/reporting system is M13 phage
• Fusion of target proteins to either of the surface
proteins g3p, g8p
• Propagated phages (in E.coli) express target
proteins on their surface
• A phage library contains ~108 different targets
Phage display - screening
• Immobilize bait protein on
magnetic beads
• Phages displaying target
bind to the bait
• Bound phages are eluted
and amplified in E. coli.
• Sequence phage DNA to
determine target protein that
interacted with given bait.