Covalent\nAttachment of Functional Proteins to Microfiber\nSurfaces via a General Strategy for Site-Selective Tetrazine Ligation

Surface\nmodification of materials with proteins has various biological\napplications, and hence the methodology for surface modification needs\nto accommodate a wide range of proteins that differ in structure,\nsize, and function. Presented here is a methodology that uses the\nAffinity Bioorthogonal Chemistry (ABC) tag, 3-(2-pyridyl)-6-methyltetrazine\n(PyTz), for the site-selective modification and purification of proteins\nand subsequent attachment of the protein to <i>trans</i>-cyclooctene (TCO)-functionalized hydrogel microfibers. This method\nof surface modification is shown to maintain the functionality of\nthe protein after conjugation with proteins of varying size and functionalities,\nnamely, HaloTag, NanoLuc luciferase (NanoLuc), and fibronectin type\nIII domains 9–10 (FNIII 9–10). The method also supports\nsurface modification with multiple proteins, which is shown by the\nsimultaneous conjugation of HaloTag and NanoLuc on the microfiber\nsurface. The ability to control the relative concentrations of multiple\nproteins presented on the surface is shown with the use of HaloTag\nand superfolder GFP (sfGFP). This application of the ABC-tagging methodology\nexpands on existing surface modification methods and provides flexibility\nin the site-selective protein conjugation methods used along with\nthe rapid kinetics of tetrazine ligation.