A strategy for high-throughput screening of ligands suitable for molecular imprinting of proteins

For facilitating the identification of appropriate functionalities that may serve as a binding motif of functional monomers, a selection strategy based on high-throughput screening of the binding properties of readily available sorbent materials has been developed. Thereby, the affinity of such ligands to the protein of interest may be rapidly determined. From these studies, it is anticipated that ligand functionalities will be derived, which may lead to advanced selection and design of dedicated functional monomers suitable for decorating the surface of a scavenger material. Thus, specific binding of the target protein of interest should be enabled even in complex solutions such as e.g., biotechnologically relevant cell lysates.In the present contribution, an automated screening method for studying ligand interactions of selected sorbent materials with pepsin – a protein of the protease family – was developed. Aqueous buffer solutions containing pepsin at known constant concentration were pipetted through an array of miniaturized chromatographic solid phase extraction (SPE) columns containing a variety of sorbent materials, and the eluted solutions were analyzed by UV/vis spectroscopy. The established screening protocol was validated against resin materials of known interaction with pepsin. Finally, the developed screening strategy was adapted for a robot system enabling high-throughput screening for a wide variety of sorbent materials and ligand functionalities in a fully automated approach.The obtained results clearly indicate that the established screening routine provides valuable data for characterizing resin-immobilized ligands, and their affinity toward pepsin.

► High-throughput screening method for pepsin and its binding affinity toward resin-immobilized ligands developed and tested. ► Reproducible robot screening strategy developed with automated UV spectroscopic analysis of binding properties. ► Screening routine applicable to the characterization of ligands and of already synthesized molecular imprinted polymers.