Two-point immobilization of a conformation-specific beta2-adrenoceptor for recognizing the receptor agonists or antagonists inspired by binding-induced DNA assembly
Two-point immobilization of a conformation-specific beta2-adrenoceptor for recognizing the receptor agonists or antagonists inspired by binding-induced DNA assembly
复制标题
DOI：
10.1039/d1bm01222c
复制
发表时间：
2021-10-08
影响因子：
6.6
通讯作者：
Zhao, Xinfeng
中科院分区：
文献类型：
--
作者：
Gao, Juan;Yuan, Xinyi;Zhao, Xinfeng
固定化蛋白在许多领域都取得了进展，比如药物发现。虽然这一领域在过去的三十年中迅速发展，但G蛋白偶联受体(GPCR)的固定化平台仍然没有前景，因为它在当前方法相对苛刻的条件下不稳定。以β(2)-肾上腺素能受体(beta(2)-AR)为例，我们提出了一种结合His(6)标签捕获系统、构象特异性适配子和靶向结合诱导的DNA杂交来固定化GPCRs的一般策略。通过多种分析手段对微球的形态进行了表征，证实了微球表面存在单层β(2)-AR。放射配基结合实验和免疫透射电子显微镜显示了良好的配基和抗体结合活性。一个使用固定化受体作为固定相的层析案例研究表明，具有明显的构象特异性，能够选择性地识别受体激动剂或拮抗剂。由于固定化过程中的竞争性链置换，该方法能够灵敏地直接测定表面受体密度，这对大多数已报道的检测方法提出了极大的挑战。这种方法有可能成为一种固定具有特定方向、构象、功能和密度的GPCRs的通用策略，从而为准确实现受体-配体结合作用和高效筛选受体激动剂或拮抗剂铺平道路。
Immobilized protein has advanced in many areas like drug discovery. While this field evolved rapidly over the last three decades, the immobilization platform for the G-protein-coupled receptor (GPCR) remains unpromising due to its instability under the relatively harsh conditions of current methodologies. Taking beta(2)-adrenoceptor (beta(2)-AR) as an example, we presented here a general strategy for immobilization of GPCRs by combining the His(6)-tag trap system, conformation-specific aptamer, and target binding induced DNA hybridization. Morphology characterization by diverse assays confirmed a monolayer of beta(2)-AR on the microsphere surface. The radio-ligand binding assay and immuno-transmission electron microscopy showed desirable ligand- and antibody-binding activities. A case study of chromatography using the immobilized receptor as a stationary phase exhibited a demonstrable conformation specificity that enables the selective recognition of the receptor agonists or antagonists. Owing to the competitive strand displacement during the immobilization, the method proved to be capable of sensitively and directly determining the receptor density on the surface which enormously challenges most of the reported assays. This method is possible to turn into a general strategy for the immobilization of GPCRs with a defined orientation, conformation, function, and density, thus paving the way for precisely realizing the receptor-ligand binding interaction and screening the receptor agonist or antagonist with high efficiency.