1. Gold labelling of a green fluorescent protein (GFP)-tag inside cells using recombinant nanobodies conjugated to 2.4 nm thiolate-coated gold nanoparticles
- Author
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Dris Ihiawakrim, Nadja Groysbeck, Anne Marie Haeberlé, Mariel Donzeau, Mounib Bahri, Ovidiu Ersen, Audrey Stoessel, Patrick Schultz, Guy Zuber, Stéphane Ory, Danièle Spehner, Institut des Neurosciences Cellulaires et Intégratives (INCI), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), schultz, patrick, Biotechnologie et signalisation cellulaire (BSC), Université de Strasbourg (UNISTRA)-Institut de recherche de l'Ecole de biotechnologie de Strasbourg (IREBS)-Centre National de la Recherche Scientifique (CNRS), Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de Physique et Chimie des Matériaux de Strasbourg (IPCMS), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et Nanosciences Grand-Est (MNGE), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, and Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
[SDV.BIO]Life Sciences [q-bio]/Biotechnology ,[SDV]Life Sciences [q-bio] ,Bioengineering ,Peptide ,02 engineering and technology ,Conjugated system ,Green fluorescent protein ,law.invention ,03 medical and health sciences ,law ,General Materials Science ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Chemistry ,General Engineering ,Sciences du Vivant [q-bio]/Biotechnologies ,General Chemistry ,Immunogold labelling ,021001 nanoscience & nanotechnology ,Heterotetramer ,Atomic and Molecular Physics, and Optics ,[SDV] Life Sciences [q-bio] ,Colloidal gold ,Recombinant DNA ,PEGylation ,Biophysics ,0210 nano-technology - Abstract
The advances in the 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 AuNP remains challenging in comparison to the conjugation of 150 kDa IgG to AuNP. In here, 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 via thiolate exchange. After pegylation, the modified AuNPs are able to non- covalently anchor Nb dimers and the subsequent complexes demonstrate the ability to 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.
- Published
- 2021
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