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.

由于生物样品中生物活性成分的复杂性，靶蛋白的筛选是一个复杂的过程。本文提出了一种基于 SpyTag/SpyCatcher (ST/SC) 介导的锚定指导蛋白质固定在二氧化硅磁珠上用于配体钓鱼的可行策略。使用 1-乙基-3-(3-二甲基氨基丙基) 碳二亚胺盐酸盐/ N将二氧化硅包覆的磁珠 (SMB) 表面上的羧基官能团与 SC 偶联-羟基磺基琥珀酰亚胺法，命名为 SC-SMBs。作为捕获蛋白模型的绿色荧光蛋白 (GFP) 被 ST 标记，并通过 ST/SC 自连接直接从相关细胞裂解物中以特定方向锚定到 SC-SMB 表面。通过电子显微镜、能量色散光谱和傅里叶变换红外光谱研究了 SC-SMB 的特性。这种独特反应的自发性和位点特异性通过电泳和荧光分析得到证实。尽管 ST-GFP 连接的 SC-SMB 的碱性稳定性并不理想，但在酸性条件（0.05 M 甘氨酸-HCl 缓冲液，pH 1-6）下，在 20% 乙醇溶液 7 内，形成的异肽键在 2 小时内是牢不可破的天，在大多数温度下。因此，我们，