Modular Site-Specific Conjugation of Nanobodies Using Two Co-Associating Tags

• Published in: International journal of molecular sciences 2022-11, Vol.23 (22), p.14405
• Main Authors: Moeglin, Eric, Barret, Lina, Chatton, Bruno, Donzeau, Mariel
• Format: Article
• Language: English
• Subjects: Antibodies; Binding; Biochemistry, Molecular Biology; Chemical Phenomena; Chromatography; Confocal microscopy; Conjugation; Coupling methods; Cytometry; E coli; Enzymes; Epidermal growth factor; ErbB-2 protein; Flow cytometry; Fluorescent indicators; Fluorophores; Genetic engineering; Growth factors; Humans; Labeling; Life Sciences; Microscopy; Modular engineering; Molecular Probes; Nanobodies; p53 Protein; Peptides; Proteins; Single-Domain Antibodies; Tags
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms232214405

The homogeneous labeling of antibodies and their fragments is a critical step for the generation of robust probes used in immuno-detection applications. To date, numerous chemical, genetic and peptide-based site-specific coupling methods have been developed. Among these methods, co-assembling peptide-tags is one of the most straightforward and versatile solutions. Here, we describe site-specific labeling of nanobodies through the use of two co-associating peptides tags, E3 and K3, originating from the tetramerization domain of p53. These E3 and K3-tags provide a simple and robust method for associating stoichiometric amount of V H and fluorescent probes, either fluorescent proteins or fluorochromes, at specific positions. As a proof of concept, a nanobody targeting the human epidermal growth factor receptor 2 (HER2), the nano-HER2 was genetically fused to the E3 and associated with different fluorescent K3-derivates. Entities were produced separately in in soluble forms at high yields and co-assembled in vitro. These molecular probes present high binding specificity on HER2-overexpressing cells in flow-cytometry with relative binding constants in the low nanomolar range and are stable enough to stain HER2-receptor on living cells followed detection using fluorescent confocal microscopy. Altogether, our results demonstrate that the non-covalent conjugation method using these two co-associating peptides can be easily implemented for the modular engineering of molecular probes for cell immuno-staining.