Cross-Linked Reticular Magnetic Beads Immobilizing Streptavidin for Fishing the Protein of Interest with High Sensitivity

Magnetic beads immobilized with affinity ligands act as a potent tool to fish the protein of interest (POI) interacting with bioactive compounds, proteins, or nucleic acids. The high loading capacity of affinity ligands is crucial to assay sensitivity. Herein, we report a simply fabricated cross-linked carboxylic magnetic bead, Fe 3 O 4 @poly(acrylic acid- co -divinylbenzene) (PAA- co -DVB), with a reticular network, exhibiting excellent magnetic response, high carboxylic functionality, and a fast reaction rate during amidation. Streptavidin (SA) is immobilized onto Fe 3 O 4 @PAA- co -DVB through the encapsulation of appropriate pores, followed by covalent binding. The obtained Fe 3 O 4 @PAA- co -DVB@SA displays a high biotin-binding capacity of 5347 pmol mg –1 and low nonspecific protein absorption with blocking. Fe 3 O 4 @PAA- co -DVB@SA shows 1.65- and 1.55-fold higher sensitivity than commercial beads and non-cross-linked beads when fishing biotinylated proteins from pig liver lysates. The mode of immobilization was further studied, and control experiments revealed that the reticular network of Fe 3 O 4 @PAA- co -DVB is indeed capable of encapsulating proteins, and the following amidation stabilizes the immobilization. Therefore, Fe 3 O 4 @PAA- co -DVB@SA is applied to two practical cases of identifying protein–protein interaction and small molecule–protein interaction. TurboID identification of suppressor-of-G2-allele-of-skp1 (SGT1) interacting protein heat shock proteins (HSP90) in N. benthamiana reveals that the POI band enriched with Fe 3 O 4 @PAA- co -DVB@SA is 7-fold denser than that of the positive control. Further verification of in vitro interaction of Mps1 ( M. oryzae mitogen-activated protein kinase I) with compound A378–0, an ATP-competitive inhibitor of kinase, demonstrates a comprehensive understanding of the action mechanism and binding affinity between proteins and their interacting molecules, and shows the feasibility and sensitivity of Fe 3 O 4 @PAA- co -DVB@SA in fishing the targeted protein for universal purposes. The excellent performance in application also offers the potential of Fe 3 O 4 @PAA- co -DVB to immobilize other affinity ligands and boost its further large-scale industrial production.