For biomedical applications, proteins may require conjugation to small and large molecules. Typical examples are dyes for imaging, cytotoxic effector molecules for cell killing, or half-life extension modules for optimized pharmacokinetics. Although many conjugation strategies are straightforward to apply, most of them do not enable site-specific and orthogonal conjugation, and do not yield a defined stoichiometry. Moreover, techniques offering these desirable features often rely on complex expression procedures and suffer from low production yields. A more promising manufacturing strategy for flexible, site-specific and stoichiometrically defined payloading of proteins is the combination of click chemistry and thiol-maleimide conjugation, which even enables dual labeling when used consecutively. Here, we describe as an example the production of Designed Ankyrin Repeat Proteins (DARPins), a non-IgG binding scaffold, in a specific E. coli strain to obtain high yields of protein carrying both a thiol and an azide group. We provide straightforward protocols for strain-promoted azide-alkyne cycloaddition (SPAAC) and thiol-maleimide conjugation, and furthermore compare these conjugation chemistries with existing alternatives like copper-catalyzed azide-alkyne cycloaddition (CuAAC). Finally, detailed instructions for reactivity analysis and yield estimations of the reactions are provided.      «

For biomedical applications, proteins may require conjugation to small and large molecules. Typical examples are dyes for imaging, cytotoxic effector molecules for cell killing, or half-life extension modules for optimized pharmacokinetics. Although many conjugation strategies are straightforward to apply, most of them do not enable site-specific and orthogonal conjugation, and do not yield a defined stoichiometry. Moreover, techniques offering these desirable features often rely on complex expr...     »