CF2H: a cell-free two-hybrid platform for rapid protein binder screening

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Protein binders are crucial for modulating biological targets in medicine.
• Developing efficient screening methods is essential for advancing protein binder discovery.
• De novo protein design offers new possibilities for creating novel binders.

Purpose of the Study:

• To introduce Cell-Free Two-Hybrid (CF2H), a novel, rapid, and sensitive method for detecting high-affinity protein-protein interactions (PPI).
• To demonstrate CF2H's utility as a screening platform for various protein binders and its applicability in biomarker detection and small-molecule modulator characterization.

Main Methods:

• CF2H utilizes a dimerization-activated DNA binding domain fused to bait and prey proteins to induce transcription upon PPI.
• The method bypasses the need for cloning, protein purification, and specialized laboratory equipment.
• CF2H was benchmarked using known Mdm2 binders and for discovering new PD-L1 binders.

Main Results:

• CF2H successfully detected interactions involving diverse protein binders, including single-domain antibodies, DARPins, and de novo designed binders.
• High-affinity binders for Mdm2 were validated, and novel high-affinity binders for PD-L1 were identified in under 24 hours.
• The study demonstrated CF2H's capability to characterize small-molecule modulators of PPI and detect protein biomarkers.

Conclusions:

• CF2H is a rapid, cost-effective, and accessible method for screening protein binders and detecting PPI.
• This cell-free system accelerates the identification of high-affinity binders for therapeutic targets like PD-L1.
• CF2H has the potential to enable a new generation of cell-free biosensors for various biomedical applications.