Protein immobilization is particularly significant in proteomics, interactomics, and in vitro drug screening. It is an essential primary step for numerous biological techniques that rely on immobilized proteins with controlled orientation, high conformational stability, and high activity (CHH). These have challenged the current immobilization strategy and demanded increasing efforts for an efficient method to meet the CHH immobilization in a single step. Herein, we proposed a covalent inhibitor-based, one-step method for G protein-coupled receptor (GPCR) immobilization inspired by the covalent reaction between an epidermal growth factor receptor (EGFR)-tag and its inhibitor ibrutinib. We immobilized endothelin receptor A (ETA) containing a fusion EGFR tag onto an ibrutinib-coated macroporous silica gel. The immobilized ETA proved to have demonstrable ligand-binding activity and specificity, thus resulting in a chromatographic technology allowing receptor–ligand interaction analysis and lead identification. Such immobilization method is attractable, owing to the properties of mild reacting conditions, fast rate, high yield, and good stability of the conjugated protein. It will be applicable to biochips, biosensors, and biocatalysts.

中文翻译：

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基于共价抑制剂的内皮素A固定化一步法：从配体识别到潜在顾客识别。

蛋白质固定化在蛋白质组学，相互作用组学和体外药物筛选中尤其重要。对于依赖于具有受控方向，高构象稳定性和高活性（CHH）的固定化蛋白质的众多生物学技术而言，这是必不可少的第一步。这些挑战了当前的固定化策略，并要求加大努力以一种有效的方法来一步实现CHH固定化。在本文中，我们提出了一种基于共价抑制剂的一步法进行G蛋白偶联受体（GPCR）固定的方法，该方法受表皮生长因子受体（EGFR）标签与其抑制剂依鲁替尼之间的共价反应的启发。我们将含有融合EGFR标签的内皮素受体A（ETA）固定在依鲁替尼涂层的大孔硅胶上。固定化的ETA被证明具有可证明的配体结合活性和特异性，因此产生了一种色谱技术，可进行受体-配体相互作用分析和前导识别。由于温和的反应条件，快速的速率，高的收率和结合蛋白的良好稳定性，这种固定方法是吸引人的。它将适用于生物芯片，生物传感器和生物催化剂。