Enhancing Chromatographic Performance of Immobilized Angiotensin II Type 1 Receptor by Strain-Promoted Alkyne Azide Cycloaddition through Genetically Encoded Unnatural Amino Acid.
Enhancing Chromatographic Performance of Immobilized Angiotensin II Type 1 Receptor by Strain-Promoted Alkyne Azide Cycloaddition through Genetically Encoded Unnatural Amino Acid.
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DOI：
10.1021/acs.analchem.2c03130
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发表时间：
2022-11
影响因子：
7.4
通讯作者：
Haiyue Zuo;Ting Li;Dandan Zhang;Jing Ma;Zilong Zhang;Yu-Shan Ou;Xiaojuan Lian;Jiatai Yin;Qian Li;Xinfeng Zhao
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文献类型：
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作者：
Haiyue Zuo;Ting Li;Dandan Zhang;Jing Ma;Zilong Zhang;Yu-Shan Ou;Xiaojuan Lian;Jiatai Yin;Qian Li;Xinfeng Zhao
在整合到固体表面的过程中，为生物正交反应引入的标签对蛋白质活性的影响很少得到研究。这代表了提高固定化蛋白检测性能的主要挑战。本文通过基因编码叠氮-炔环加成反应、Halo-和snap -标记生物正交反应合成固定化血管紧张素II型1受体（AT1R）测定色谱性能，实现了融合标签与其反应效率之间的关系。我们证明，在受体中掺入非天然氨基酸的固定化可以最大限度地减少厄贝沙坦、非马沙坦、氯沙坦和塔沙坦的峰尾和宽度，而通过大标签（SNAP和Halo）的附着会导致严重的不对称峰。在第一次固定时，四种药物与AT1R的结合常数似乎比其他两种附着物大1个数量级。这种增强可能是由于小标签提高了缔合速率常数和相对相同的解离速率。通过基因编码叠氮化物-炔反应制备的固定化AT1R在提高色谱性能的同时，被用于分析Uncaria Schreber noms . cons.提取物，鉴定出hynchophylline是与受体结合的特异性配体。随着固定化蛋白向着多样化的检测方向发展，我们的研究结果为融合标签及其介导检测性能的反应效率之间的关系提供了前所未有的见解，因此致力于创建蛋白质功能化表面，以精确确定药物-蛋白质相互作用并发现蛋白质的特定伙伴。
During integration to the solid surface, the effects of tags introduced for bioorthogonal reactions on protein activity have received far less investigation. This represents the major challenge of improving the performance of the immobilized protein-based assays. Herein, the relationship between the fusion tags and their reaction efficiency in mediating the assay performance was realized by determining the chromatographic performance using genetically encoded azide-alkyne cycloaddition, and Halo- and SNAP-tagged bioorthogonal reactions for synthesizing immobilized angiotensin II type 1 receptor (AT1R). We demonstrated that immobilization with the incorporation of unnatural amino acid in the receptor minimizes the peak tailings and broadenings of irbesartan, fimasartan, losartan, and tasosartan, while attachment via large tags (SNAP and Halo) leads to serious asymmetry peaks. Upon the first immobilization, the association constants of the four drugs to AT1R appeared to be 1 order of magnitude greater than the other two attachments. Such enhancement is likely reasoned by the improved association rate constants and the relatively identical dissociation rates due to the small tag. While demonstrating improved chromatographic performance, the immobilized AT1R prepared by the genetically encoded azide-alkyne reaction was applied in analyzing Uncaria Schreber nom. cons. extract, which identified hynchophylline as a specific ligand binding to the receptor. As immobilized proteins move toward diverse assays, our findings provide an unprecedented insight into the relation between fusion tags and their reaction efficiency in mediating the assay performance, which is thus dedicated to the creation of a protein-functionalized surface for precisely determining the drug-protein interaction and discovering the specific partner of the protein.