Vandevenne Marylène, Dondelinger Mathieu, Yunus Sami, Freischels Astrid, Freischels Régine, Crasson Oscar, Rhazi Noureddine, Bogaerts Pierre, Galleni Moreno, Filée Patrice
Life Science Department, University of Liège;
Life Science Department, University of Liège.
J Vis Exp. 2018 Feb 1(132):55414. doi: 10.3791/55414.
Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used β-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP β-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the β-lactamase activity. This suggests that the nanobody and β-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid β-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.
生物传感器正变得越来越重要，并在病原体检测、分子诊断、环境监测和食品安全控制等各个领域得到应用。在此背景下，我们在多项蛋白质-蛋白质相互作用研究中使用β-内酰胺酶作为高效报告酶。此外，它们接受肽或结构化蛋白质/结构域插入的能力极大地促进了利用这些酶来生成嵌合蛋白。在最近的一项研究中，我们将一个单域抗体片段插入地衣芽孢杆菌BlaPβ-内酰胺酶中。这些小结构域，也称为纳米抗体，被定义为骆驼科动物单链抗体的抗原结合结构域。与常见的双链抗体一样，它们对其靶标表现出高亲和力和特异性。所得嵌合蛋白对其靶标表现出高亲和力，同时保留β-内酰胺酶活性。这表明纳米抗体和β-内酰胺酶部分仍然具有功能。在本工作中，我们报告了一种详细方案，该方案将我们的混合β-内酰胺酶系统与生物传感器技术相结合。由于对酶催化活性释放的质子进行电导测量，纳米抗体与其靶标的特异性结合可以被检测到。
