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Title: | Site-selective functionalization of nanobodies using intein-mediated protein ligation for innovative bioconjugation | Authors: | GRAULUS, Geert-Jan TA, Duy Tien TRAN, Huong HANSEN, Rebekka BILLEN, Brecht ROYACKERS, Erik NOBEN, Jean-Paul Devoogdt, Nick Muyldermans, Serge GUEDENS, Wanda ADRIAENSENS, Peter |
Issue Date: | 2019 | Publisher: | SPRINGER INTERNATIONAL PUBLISHING AG | Source: | Walker, John M.; Massa, Sam; Devoogdt, Nick (Ed.). Bioconjugation: Methods and Protocols, SPRINGER INTERNATIONAL PUBLISHING AG, | Series/Report: | Methods in Molecular Biology | Abstract: | An expression strategy is presented in order to produce nanobodies modified with a clickable alkyne function 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 towards the nanobody targeted antigen. The presented protocol benefits from time- and cost-effectiveness and allows a feasible up-scaling of functionalized (here alkynated) nanobodies. The production of high quantities of site-specifically modified nanobodies paves the way to i) novel biosurface applications that demand for homogeneously oriented nanobodies having their active site fully accessible for target (e.g. biomarker) binding, and ii) 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. | Keywords: | Protein functionalization; intein; nanobody; click chemistry | Document URI: | http://hdl.handle.net/1942/27970 | ISBN: | 9781493996537 | Category: | B2 | Type: | Book Section | Validations: | vabb 2021 |
Appears in Collections: | Research publications |
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2018Jul19 Guedens_revised (1).pdf Restricted Access | Peer-reviewed author version | 213.78 kB | Adobe PDF | View/Open Request a copy |
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