Towards multiplexed protein-protein interaction analysis using protein tag-specific nanobodies

Area of Science:

• Biochemistry and Molecular Biology
• Cellular Biology
• Proteomics

Background:

• Dynamic protein-protein interactions (PPIs) are crucial for cellular functions and disease development.
• Current bead-based assays often use bacterial-derived proteins, limiting their biological relevance due to lack of proper folding and modifications.
• Developing robust methods for studying PPIs is essential for understanding cellular processes and identifying therapeutic targets.

Purpose of the Study:

• To develop a novel bead-based protein array method for studying dynamic protein-protein interactions (PPIs).
• To enable the generation of protein arrays using purified bait-proteins from both bacterial and mammalian expression systems.
• To demonstrate the utility of this method in analyzing complex signaling pathways, such as the Wnt pathway.

Main Methods:

• Expression of bait-proteins as GST- or GFP-fusion constructs in bacterial and mammalian cells.
• Purification and site-directed immobilization of bait-proteins from crude lysates using tag-specific nanobodies coupled to color-coded beads.
• Generation of miniaturized, multiplexed bead-based protein arrays for pulldown studies.

Main Results:

• Successful isolation of highly pure and stable bait-proteins from small-scale expression cultures.
• Demonstration of dynamic changes in endogenous β-catenin following proteasomal inhibition or signaling pathway perturbation.
• Proof-of-principle analysis of Wnt-pathway activation using multiplexed μ-scale pulldowns.

Conclusions:

• The developed method allows for the generation of bead-based protein arrays with numerous bait-proteins from diverse expression systems.
• Utilizing eukaryotic expression systems enhances the stability and binding properties of bait-proteins for PPI studies.
• This approach facilitates the study of complex PPIs and cellular signaling dynamics, offering advantages over traditional methods.