A preparation strategy for protein-oriented immobilized silica magnetic beads with Spy chemistry for ligand fishing

Due to the complexity of bioactive ingredients in biological samples, the screening of target proteins is a complex process. Herein, a feasible strategy for directing protein immobilization on silica magnetic beads for ligand fishing based on SpyTag/SpyCatcher (ST/SC)-mediated anchoring is presented. Carboxyl functional groups on the surface of silica-coated magnetic beads (SMBs) were coupled with SC using the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysulfosuccinimide method, named SC-SMBs. The green fluorescent protein (GFP), as the capturing protein model, was ST-labeled and anchored at a specific orientation onto the surface of SC-SMBs directly from relevant cell lysates via ST/SC self-ligation. The characteristics of the SC-SMBs were studied via electron microscopy, energy dispersive spectroscopy, and Fourier transform infrared spectroscopy. The spontaneity and site-specificity of this unique reaction were confirmed via electrophoresis and fluorescence analyses. Although the alkaline stability of ST-GFP-ligated SC-SMBs was not ideal, the formed isopeptide bond was unbreakable under acidic conditions (0.05 M glycine-HCl buffer, pH 1–6) for 2 h, under 20% ethanol solution within 7 days, and at most temperatures. We, therefore, present a simple and universal strategy for the preparation of diverse protein-functionalized SMBs for ligand fishing, prompting its usage on drug screening and target finding.