Covalent Attachment of Functional Proteins to Microfiber Surfaces via a General Strategy for Site-Selective Tetrazine Ligation

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