Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 23, Issue 22, Pages -Publisher
MDPI
DOI: 10.3390/ijms232214405
Keywords
VHH; nanobody; conjugation; site-specific
Funding
- Centre National de la Recherche Scientifique
- University of Strasbourg
- French Ministry of Research
- Ligue Regionale contre le Cancer (CCIR-GE)
- Fondation ARC pour la recherche sur le cancer
- ITI Innovec (IdEx) [ANR-10-IDEX-0002]
- SFRI [ANR-20-SFRI-0012]
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This study describes a method for site-specific labeling of nanobodies using two co-assembling peptide tags, E3 and K3. The method provides a simple and robust approach for associating VHH and fluorescent probes, and demonstrates the potential application of these molecular probes in cell immuno-staining.
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 VHH 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 Escherichia coli 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.
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