Groysbeck, Nadja, Donzeau, Mariel, Stoessel, Audrey, Haeberle, Anne-Marie, Ory, Stephane, Spehner, Daniele, Schultz, Patrick, Ersen, Ovidiu, Bahri, Mounib, Ihiawakrim, Dris et al (show 1 more authors)
(2021)
Gold labelling of a green fluorescent protein (GFP)-tag inside cells using recombinant nanobodies conjugated to 2.4 nm thiolate-coated gold nanoparticles.
NANOSCALE ADVANCES, 3 (24).
pp. 6940-6948.
Abstract
Advances in microscopy technology have prompted efforts to improve the reagents required to recognize specific molecules within the intracellular environment. For high-resolution electron microscopy, conjugation of selective binders originating from the immune response arsenal to gold nanoparticles (AuNPs) as contrasting agents is the method of choice to obtain labeling tools. However, conjugation of the minimal sized 15 kDa nanobody (Nb) to AuNPs remains challenging in comparison to the conjugation of 150 kDa IgG to AuNPs. Herein, effective Nb-AuNP assemblies are built using the selective and almost irreversible non-covalent associations between two peptide sequences deriving from a p53 heterotetramer domain variant. The 15 kDa GFP-binding Nb is fused to one dimerizing motif to obtain a recombinant Nb dimer with improved avidity for GFP while the other complementing dimerizing motif is equipped with thiols and grafted to a 2.4 nm substituted thiobenzoate-coordinated AuNP <i>via</i> thiolate exchange. After pegylation, the modified AuNPs are able to non-covalently anchor Nb dimers and the subsequent complexes demonstrate the ability to form immunogold label GFP-protein fusions within various subcellular locations. These tools open an avenue for precise localization of targets at high resolution by electron microscopy.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | 3403 Macromolecular and Materials Chemistry, 34 Chemical Sciences, 40 Engineering, 4016 Materials Engineering, 4018 Nanotechnology, Nanotechnology, Bioengineering |
| Divisions: | Faculty of Science and Engineering > School of Engineering |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 20 Dec 2021 15:19 |
| Last Modified: | 16 Aug 2024 12:22 |
| DOI: | 10.1039/d1na00256b |
| Open Access URL: | https://pubs.rsc.org/en/content/articlelanding/202... |
| Related URLs: | |
| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3145700 |
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