Crasson O, Rhazi N, Jacquin O, Freichels A, Jérôme C, Ruth N, Galleni M, Filée P, Vandevenne M
Macromolécules Biologiques, Center D'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart-Tilman, Liège B4000, Belgium.
Chimie des Macromolécules et des Matériaux Organiques (CERM), Institut de Chimie B6a, Université de Liège, Sart-Tilman, Liège B4000, Belgium.
Protein Eng Des Sel. 2015 Oct;28(10):451-60. doi: 10.1093/protein/gzv020. Epub 2015 Apr 6.
Antibody-based products constitute one of the most attractive biological molecules for diagnostic, medical imagery and therapeutic purposes with very few side effects. Their development has become a major priority of biotech and pharmaceutical industries. Recently, a growing number of modified antibody-based products have emerged including fragments, multi-specific and conjugate antibodies. In this study, using protein engineering, we have functionalized the anti-hen egg-white lysozyme (HEWL) camelid VHH antibody fragment (cAb-Lys3), by insertion into a solvent-exposed loop of the Bacillus licheniformis β-lactamase BlaP. We showed that the generated hybrid protein conserved its enzymatic activity while the displayed nanobody retains its ability to inhibit HEWL with a nanomolar affinity range. Then, we successfully implemented the functionalized cAb-Lys3 in enzyme-linked immunosorbent assay, potentiometric biosensor and drug screening assays. The hybrid protein was also expressed on the surface of phage particles and, in this context, was able to interact specifically with HEWL while the β-lactamase activity was used to monitor phage interactions. Finally, using thrombin-cleavage sites surrounding the permissive insertion site in the β-lactamase, we reported an expression system in which the nanobody can be easily separated from its carrier protein. Altogether, our study shows that insertion into the BlaP β-lactamase constitutes a suitable technology to functionalize nanobodies and allows the creation of versatile tools that can be used in innovative biotechnological assays.
基于抗体的产品是用于诊断、医学成像和治疗目的的最具吸引力的生物分子之一，副作用极少。它们的开发已成为生物技术和制药行业的主要优先事项。最近，出现了越来越多基于修饰抗体的产品，包括片段、多特异性抗体和偶联抗体。在本研究中，我们利用蛋白质工程技术，通过将抗鸡蛋清溶菌酶（HEWL）的骆驼科VHH抗体片段（cAb-Lys3）插入地衣芽孢杆菌β-内酰胺酶BlaP暴露于溶剂的环中，使其功能化。我们表明，生成的杂合蛋白保留了其酶活性，而展示的纳米抗体保留了以纳摩尔亲和力范围抑制HEWL的能力。然后，我们成功地将功能化的cAb-Lys3应用于酶联免疫吸附测定、电位生物传感器和药物筛选测定中。杂合蛋白也在噬菌体颗粒表面表达，在这种情况下，它能够与HEWL特异性相互作用，同时利用β-内酰胺酶活性监测噬菌体相互作用。最后，利用β-内酰胺酶中允许插入位点周围的凝血酶切割位点，我们报道了一种表达系统，其中纳米抗体可以很容易地从其载体蛋白中分离出来。总之，我们的研究表明，插入BlaPβ-内酰胺酶构成了一种使纳米抗体功能化的合适技术，并允许创建可用于创新生物技术测定的通用工具。
