基于β-酶的电导生物传感器检测技术在分子间相互作用中的应用
Summary
在这项工作中, 我们报告了一个新的方法来研究蛋白质-蛋白质相互作用的电导生物传感器的基础上的混合β-酶技术。这种方法依赖于质子在β-内酰胺水解时的释放。
Abstract
生物传感器在病原检测、分子诊断、环境监测、食品安全控制等各个领域的应用越来越重要。在这方面, 我们使用β-酶作为有效的报告酶在几个蛋白质-蛋白质相互作用研究。此外, 他们接受多肽或结构蛋白/域插入的能力强烈鼓励使用这些酶产生嵌合蛋白。在最近的一项研究中, 我们在芽孢杆菌地衣BlaP β-酶中插入了一个单域抗体片段。这些小的域, 也称为 nanobodies, 被定义为骆驼单链抗体的抗原结合域。就像常见的双链抗体一样, 它们具有很高的亲和力和特异的靶向。所产生的嵌合蛋白在保持β-酶活性的同时, 对其靶向性具有较高的亲和力。这表明, nanobody 和β-酶基保持功能。在目前的工作中, 我们报告了一个详细的协议, 结合我们的混合β-酶系统的生物传感器技术。由于酶的催化活性释放出的质子的电导测量, 可以检测到 nanobody 对其目标的特定约束力。
Introduction
生物传感器是一种与物理或化学信号装置相结合的分析设备, 称为传感器1。然后, 可以对记录的信号进行解释和转换, 以监测固定和自由伙伴之间的相互作用。大多数的生物传感器涉及使用抗体来检测分析如激素或不同的病原体标记物2。可以使用不同的传感器格式, 包括基于质量的、磁的、光学的或电化学的生物传感器。后者是最常用的传感器之一, 通过将绑定事件转换为电信号来发挥作用。所有基于抗体的生物传感器的性能和灵敏度主要取决于两个参数: i) 抗体的质量和 ii) 用于生成信号2的系统的属性。
抗体是由两个重链和两个光链组成的高分子质量二蛋白 (150–160 kDa)。光与重链之间的相互作用主要通过疏水相互作用以及一个守恒的二硫化键稳定。每个链包括一个变量域, 它通过三变区域 (CDR1-2-3) 来与抗原相互作用。尽管在该领域取得了许多进展, 但具有低成本表达系统 (例如、大肠杆菌) 的全长抗体的大规模表达往往导致不稳定和聚合蛋白的产生。这就是为什么各种各样的抗体片段被设计了例如单链可变片段3 (ScFvs ≈ 25 kDa)。它们由一个重和一个轻链的可变域组成, 由一个合成氨基酸序列共价键连接。然而, 这些碎片往往表现出很差的稳定性, 并有聚合的倾向, 因为它们将其疏水区域的很大一部分暴露给溶剂4。在这种情况下, 单链骆驼抗体片段, 称为 nanobodies 或 VHHs, 似乎是很好的替代 ScFvs。这些域对应于骆驼单链抗体的可变域。与常规抗体相比, 骆驼抗体缺乏光链, 只包含两个重链5。因此, nanobodies 是最小的单体抗体片段 (12 kDa) 能够绑定到一个抗原的亲和力类似于常规抗体6。此外, 与其他全长抗体或抗体片段相比, 它们具有更好的稳定性和溶解度。最后, 他们的小尺寸和他们的扩展 CDR3 循环允许他们辨认隐晦的表位并且束缚到酵素活跃站点7,8。目前, 这些领域得到了相当的重视, 并已与生物传感器技术相结合。例如, 黄等人。开发了一种基于 nanobody 的生物传感器, 用于检测和量化人类前列腺特异抗原 (PSA)9。
如上所述, 生物传感器检测的一个重要参数是用于产生电信号的系统的效率。因此, 基于酶的电化学生物传感器越来越受到人们的关注, 并被广泛应用于医疗保健、食品安全和环境监测等各种领域。这些生物传感器依赖于一种酶对基质的催化水解来产生电信号。在这种情况下, β-酶被证明是更具体, 更敏感和更容易实施实验比其他许多酶, 如碱性磷酸酶或辣根过氧化物酶10。β-酶是酶, 负责对β内酰胺抗生素的细菌耐药性, 通过水解它们。它们单体, 非常稳定, 高效, 体积小。此外, 域/肽插入β-酶产生双功能嵌合蛋白, 被证明是研究蛋白质-配体相互作用的有效工具。事实上, 最近的研究表明, 在 TEM1 β-酶中插入抗体可变片段的结果是嵌合蛋白, 仍然能够与其靶抗原的高度亲和性结合。有趣的是, 抗原结合被证明能诱发 TEM1 催化活性的构调节11,12。此外, 我们在几项研究中表明, 蛋白质域插入到一个允许循环的芽孢杆菌地衣BlaP β-酶产生功能嵌合蛋白, 非常适合监测蛋白配体的相互作用13 ,14。我们最近在 BlaP15的这个允许插入站点中插入了一个名为 cAb-Lys3 的 nanobody。这 nanobody 被证明绑定到母鸡-蛋清溶菌酶 (HEWL) 和抑制其酶活性16。我们表明, 生成的杂交蛋白, 命名为 BlaP-cAb-Lys3, 保留了高特异性/亲和力对 HEWL, 而β-酶活性保持不变。然后, 我们成功地将混合β-酶技术与电化学生物传感器相结合, 结果表明, 产生的电信号量与固定在电极上的 BlaP-cAb-Lys3 和 HEWL 的相互作用有关。事实上, β-内酰胺抗生素的水解 BlaP 诱导质子释放, 可以转化为定量的电信号。这种组合的混合β-酶技术与电化学生物传感器是快速, 灵敏, 定量, 并允许实时测量生成的信号。本文介绍了这种方法。
Protocol
Representative Results
Discussion
在这项工作中, 我们提出了一种方法来化 nanobody 使用 BlaP β-酶作为载体蛋白, 我们表明, 我们可以成功地实现的结果混合蛋白的电位传感器检测。我们工作的主要创新方面与其他生物传感器的检测相比, 是抗体部分与产生电信号的酶活性的共价耦合。这种所谓的蛋白质插入技术带来的优势和局限性, 将是本节的主要重点。
混合β-酶技术的优势。
β-酶是高效…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者承认比利时的瓦隆地区在 SENSOTEM 和 NANOTIC 研究项目的框架内, 以及国家科研基金 (储备-f.n.r.) 的财政支持。
Materials
| Reagents | |||
| KH2PO4 | Sigma-Aldricht | V000225 | |
| K2HPO4 | Sigma-Aldricht | 1551128 | |
| NaCl | Sigma-Aldricht | S7653 | |
| Tris–HCl | Roche | 10812846001 | |
| EDTA | Sigma-Aldricht | E9884 | |
| KCl | Sigma-Aldricht | P9541 | |
| Na2HPO4 | Sigma-Aldricht | NIST2186II | |
| 2-mercaptoethanol | Sigma-Aldricht | M6250 | |
| alanine | Sigma-Aldricht | A7627 | |
| HClO4 | Fluka | 34288 | 1M HClO4 solution, distributor : Sigma-Aldricht |
| casein hydrolysate | Sigma-Aldricht | 22090 | |
| benzylpenicillin sodium | Sigma-Aldricht | B0900000 | |
| hen egg white lysozyme | Roche | 10837059001 | |
| heptane | Sigma-Aldricht | 246654 | |
| methanol | Sigma-Aldricht | 322415 | |
| ammonium hydroxide solution | Sigma-Aldricht | 380539 | 28% NH3 in H2O, purified by double-distillation (concentrated?) |
| Laboratory consumables | |||
| 6-well plate | Greiner Bio-One | 657165 | CELLSTAR 6-Well Plate |
| Equipment | |||
| pH meter | WTW | 1AA110 | Lab pH meter inoLab pH 7110 |
| vacuum and filtration system | Nalgene | NALG300-4100 | Filter holders with receiver, distributor : VWR |
| potentiometric sensor chips | manufactured by Yunus and colleagues (ref 16) | ||
| PGSTAT30 Autolab | Metrohm Autolab | discontinued, succesor Autolab PGSTAT302N | |
| digital multimeter, METRAHit 22M | Gossen Metrawatt | discontinued, successor Metrahit Base |
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