Protein-protein interactions

Why study protein interactions?•To infer function•To understand regulatory networks

ApproachWith given bait, discover target

Methods•Two-hybrid system•Phage display

Two-hybrid system -1.target plasmid

• Target plasmid directs transcription of the N-terminal 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.