Bio-nanocapsule-based scaffold improves the sensitivity and ligand-binding capacity of mammalian receptors on the sensor chip
Bio-nanocapsule-based scaffold improves the sensitivity and ligand-binding capacity of mammalian receptors on the sensor chip
复制标题
基于生物纳米胶囊的支架提高了传感器芯片上哺乳动物受体的灵敏度和配体结合能力
DOI：
10.1002/biot.201500443
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发表时间：
2016
影响因子：
4.7
通讯作者：
S.
中科院分区：
文献类型：
--
作者：
Iijima;M.;Yoshimoto;N.;Niimi;T.;Maturana;A.D.;Kuroda;S.
哺乳动物受体被认为是药物发现的靶分子，并且主要通过使用固定化受体的配体结合测定来筛选化学文库中的潜在拮抗剂和激动剂。已经开发了一种约30 nm的生物纳米胶囊（BNC），其显示蛋白A衍生的免疫球蛋白G（IgG）Fc结合Z结构域的串联形式（表示为ZZ-BNC），用于在免疫传感器的固相上聚集和定向固定IgG。在这项研究中，人IgG 1 Fc融合的血管内皮生长因子（VEGF）受体通过ZZ-BNC固定在石英晶体微天平的传感器芯片上（ZZ-BNC-涂层）。与直接吸附和蛋白A包被相比，传感器芯片显示出更高的灵敏度（分别为1.46和1.165倍）和更大的配体结合能力（分别为1.44和1.18倍）。此外，通过ZZ-BNC-包被，与其受体结合的VEGF分子数量从0.20（直接吸附）增加到2.06，强烈表明ZZ-BNC通过定向固定减少了配体识别位点附近的空间位阻。类似地，瘦素和催乳素受体的敏感性和配体结合能力都以与VEGF受体相当的水平增强。因此，ZZ-BNC和Fc融合受体的组合可以显著改善配体结合测定的功能。
Mammalian receptors are recognized as target molecules for drug discovery, and chemical libraries have been screened for both potential antagonists and agonists mainly by ligand‐binding assays using immobilized receptors. A bio‐nanocapsule (BNC) of approximately 30 nm that displays a tandem form of the protein A‐derived immunoglobulin G (IgG) Fc‐binding Z domains (denoted as ZZ‐BNC) has been developed for both clustering and oriented immobilization of IgGs on the solid phase of immunosensors. In this study, human IgG1 Fc‐fused vascular endothelial growth factor (VEGF) receptor was immobilized through ZZ‐BNC on the sensor chip of quartz crystal microbalance (ZZ‐BNC‐coating). When compared with direct adsorption and protein A‐coating, the sensor chip showed higher sensitivity (∽46‐ and ∽165‐fold, respectively) and larger ligand‐binding capacity (∽4‐ and ∽18‐fold, respectively). Furthermore, the number of VEGF molecules bound to its receptor increased from 0.20 (direct adsorption) to 2.06 by ZZ‐BNC‐coating, strongly suggesting that ZZ‐BNC reduced the steric hindrance near ligand recognition sites through oriented immobilization. Similarly, the sensitivity and ligand‐binding capacity of leptin and prolactin receptors were both enhanced at a level comparable to that observed for the VEGF receptor. Thus, the combination of ZZ‐BNC and Fc‐fused receptors could significantly improve the function of ligand‐binding assays.