Directed Covalent Immobilization of Fluorescently Labeled Cytokines

Cytokines are important mediators coordinating inflammation and wound healing in response to tissue damage and infection. Therefore, immobilization of cytokines on the surface of biomaterials is a promising approach to improve biocompatibility. Soluble cytokines signal through receptors on the cell surface leading to cell differentiation, proliferation, or other effector functions. Random immobilization of cytokines on surfaces will result in a large fraction of inactive protein due to impaired cytokine–receptor interaction. We developed a strategy that combined (i) directed covalent coupling of cytokines, (ii) quantification of coupling efficiency through fluorescence detection, and (iii) a reliable protease cleavage assay to control orientation of coupling. For this purpose, fusion proteins of the SNAP-tag followed by an enterokinase recognition site, yellow fluorescent protein (YFP), and the cytokine of interest being either interleukin-6 (IL-6) or oncostatin M (OSM) were generated. The SNAP-tag is a d...