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Site-Selective Functionalization of Nanobodies Using Intein-Mediated Protein Ligation for Innovative Bioconjugation

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Bioconjugation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2033))

Abstract

An expression strategy is presented in order to produce nanobodies modified with a clickable alkyne functionality at their C-terminus via the intein-mediated protein ligation (IPL) technique. The protocol focuses on the cytoplasmic expression and extraction of a nanobody–intein–chitin binding domain (CBD) fusion protein in E. coli SHuffle® T7 cells, in the commonly used Luria–Bertani (LB) medium. The combination of these factors results in a high yield and nearly complete alkynation of the nanobody at its C-terminus via IPL. The resulting alkynated nanobodies retain excellent binding capacity toward the nanobody targeted antigen. The presented protocol benefits from time- and cost-effectiveness and allows for a feasible upscaling of functionalized (here alkynated) nanobodies. The production of high quantities of site-specifically modified nanobodies paves the way to (1) novel biosurface applications that demand for homogeneously oriented nanobodies having their active site fully accessible for target (e.g., biomarker) binding, and (2) innovative applications such as localized drug delivery and image guided surgery by covalent “click” chemistry coupling of these alkynated nanobodies to a multitude of azide-containing counterparts as there are drug containing polymers and contrast labeling agents.

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Author information

Author notes

  1. Duy Tien Ta

    Present address: Downstream Processing Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A∗STAR), Singapore, Singapore

Authors and Affiliations

  1. Biomolecule Design Group, Institute for Materials Research (IMO), Hasselt University, Diepenbeek, Belgium

    Geert-Jan Graulus, Duy Tien Ta, Huong Tran, Rebekka Hansen, Brecht Billen, Wanda Guedens & Peter Adriaensens

  2. Biomedical Research Institute (Biomed), Hasselt University, Diepenbeek, Belgium

    Erik Royackers & Jean-Paul Noben

  3. In Vivo Cellular and Molecular Imaging Laboratory (ICMI), Vrije Universiteit Brussel, Brussels, Belgium

    Nick Devoogdt

  4. Cellular and Molecular Immunology Laboratory, Vrije Universiteit Brussel, Brussels, Belgium

    Serge Muyldermans

  5. Applied and Analytical Chemistry, Institute for Materials Research (IMO), Hasselt University, Diepenbeek, Belgium

    Peter Adriaensens

Authors
  1. Geert-Jan Graulus
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  2. Duy Tien Ta
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  3. Huong Tran
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  4. Rebekka Hansen
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  5. Brecht Billen
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  6. Erik Royackers
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  7. Jean-Paul Noben
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  8. Nick Devoogdt
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  9. Serge Muyldermans
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  10. Wanda Guedens
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  11. Peter Adriaensens
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Corresponding author

Correspondence to Peter Adriaensens .

Editor information

Editors and Affiliations

  1. Cellular and Molecular Immunology Laboratory, Vrije Universiteit Brussel, Brussels, Belgium

    Sam Massa

  2. In Vivo Cellular and Molecular Imaging Laboratory (ICMI), Vrije Universiteit Brussel, Brussels, Belgium

    Nick Devoogdt

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Graulus, GJ. et al. (2019). Site-Selective Functionalization of Nanobodies Using Intein-Mediated Protein Ligation for Innovative Bioconjugation. In: Massa, S., Devoogdt, N. (eds) Bioconjugation. Methods in Molecular Biology, vol 2033. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9654-4_9

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  • DOI: https://doi.org/10.1007/978-1-4939-9654-4_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9653-7

  • Online ISBN: 978-1-4939-9654-4

  • eBook Packages: Springer Protocols

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