Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS)

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Engineering

Background:

• Ligand-binding protein discovery is crucial for diagnostics and therapeutics.
• Existing screening methods like phage panning can be time-consuming and may miss certain protein binders.
• A need exists for rapid, efficient, and "display-less" screening technologies.

Purpose of the Study:

• To introduce and validate Periplasmic Expression with Cytometric Screening (PECS) as a novel technology.
• To demonstrate PECS's capability in isolating single-chain variable fragment (scFv) antibodies with varying affinities.
• To show that PECS can screen libraries without subcloning and identify proteins missed by conventional methods.

Main Methods:

• Utilizing Escherichia coli for periplasmic expression of protein libraries.
• Incubating cells with fluorescently labeled target ligands (up to 10 kDa).
• Employing flow cytometry to isolate cells with increased fluorescence, indicating successful binding.

Main Results:

• Successfully isolated scFv antibodies with both high and low affinities to digoxigenin.
• Demonstrated the ability to screen pre-existing phage display libraries using PECS without subcloning.
• PECS identified proteins that were not detected by traditional phage panning methods.

Conclusions:

• PECS is a powerful and rapid "display-less" technology for isolating ligand-binding proteins.
• The bacterial cell envelope acts as a dialysis bag, retaining receptor-ligand complexes.
• PECS offers advantages over conventional screening methods, including broader discovery potential.