Antibody library selection by the {beta}-lactamase protein fragment complementation assay

Area of Science:

• Biochemistry
• Molecular Biology
• Immunotechnology

Background:

• Protein fragment complementation assay (PCA) relies on reconstituting a marker protein's activity through the interaction of fused protein partners.
• This technique has broad applications in studying protein-protein interactions and screening for binding molecules.

Purpose of the Study:

• To develop and apply a beta-lactamase-based PCA for isolating single-chain Fv fragments (scFvs) targeting the human receptor RON.
• To demonstrate the efficacy of PCA in selecting functional scFvs from a naïve library.
• To utilize PCA for epitope mapping of the selected scFvs.

Main Methods:

• Genetically fusing protein partners to halves of a dissected beta-lactamase marker protein.
• Employing a naïve library and pre-selection via phage display.
• Utilizing PCA for selection of scFvs that bind to the human receptor RON.
• Screening selected scFvs against random RON fragments for epitope mapping.

Main Results:

• Successfully isolated specific scFvs that bind to the human receptor RON using a single round of PCA selection.
• Demonstrated the ability of the selected scFvs to immunoprecipitate the target receptor.
• Successfully mapped the epitopes recognized by the selected scFvs using the PCA system.

Conclusions:

• Beta-lactamase-based PCA is an effective tool for isolating functional scFvs against specific targets like the human receptor RON.
• PCA offers a streamlined approach for both scFv selection and epitope mapping, enhancing antibody discovery workflows.
• This method accelerates the identification of high-affinity antibodies with desired functional properties.