The inherent carbohydrate-binding specificities of human galectins can serve as recognition elements in both biotechnological and biomedical applications. The combination of the carbohydrate-recognition domain (CRD) of galectins fused to peptides or proteins for purification, immobilization, and imaging enables multifunctional utilization within a single protein. We present here a library of color-coded galectin fusion proteins that incorporate a His6-tag, a fluorescent protein, and a SpyCatcher or SpyTag unit to enable immobilization procedures. These galectin fusion proteins exhibit similar binding properties to the non-fused galectins with micromolar apparent binding affinities. N- and C-terminal fusion partners do not interfere with the SpyCatcher/SpyTag immobilization. By applying SpyCatcher/SpyTag-mediated SC-ST-Gal-3 conjugates, we show the stepwise formation of a three-layer ECM-like structure in vitro. Additionally, we demonstrate the SpyCatcher/SpyTag-mediated immobilization of galectins in microgels, which can serve as a transport platform for localized targeting applications. The proof of concept is provided by the galectin-mediated binding of microgels to colorectal cancer cells.

Aachen Maastricht Institute for Biobased Materials Maastricht University Brightlands Chemelot Campus Urmonderbaan 22 6167 RD Geleen The Netherlands

Department of Health Care Disciplines and Population Protection Faculty of Biomedical Engineering Czech Technical University Prague nám Sítná 3105 27201 Kladno Czech Republic

DWI Leibniz Institute for Interactive Materials e 5 Forckenbeckstr 50 52074 Aachen Germany

Functional and Interactive Polymers Institute of Technical and Macromolecular Chemistry RWTH Aachen University Worringerweg 2 52074 Aachen Germany

Laboratory for Biomaterials Institute for Biotechnology and Helmholtz Institute for Biomedical Engineering RWTH Aachen University Pauwelsstraße 20 52074 Aachen Germany

Laboratory of Biotransformation Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 Prague 4 14200 Czech Republic

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