A preparation strategy for protein-oriented immobilized silica magnetic beads with Spy chemistry for ligand fishing
Yi Yu, Jianming Hu, Shenwei Ding, Jianfeng Mei, Xudong Wang, Yanlu Zhang, Jianshu Chen, Guoqing Ying
Abstract
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.