AI Article Synopsis
- An intein-mediated protein ligation (IPL) method is introduced for producing nanobodies with a clickable alkyne group at their C-terminus, enhancing their functionality.
- The process involves expressing and extracting a nanobody-intein-chitin binding domain fusion protein in E. coli SHuffle T7 cells using LB medium, resulting in high yields of alkynated nanobodies that maintain their target-binding ability.
- This efficient and cost-effective protocol enables the large-scale production of modified nanobodies for applications in biosurfaces and advanced medical techniques like targeted drug delivery and imaging surgery through covalent coupling with azide-containing agents.
Article 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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| http://dx.doi.org/10.1007/978-1-4939-9654-4_9 | DOI Listing |
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