Functionalization of biomaterials with extra protein domains will expand their functional roles in biomedical research. The recombinant spider silk protein FN-4RepCT has been shown able to adapt various formats like coatings, nanowires, and macroscopic fibers. Functionalizing these various formats of FN-4RepCT in a site-specific manner will provide the next generation of biomaterials. The current study reports an enzymatic (sortase A) coupling method to site-specifically functionalize various formats of FN-4RepCT with target proteins. The approach is demonstrated with three different functional proteins: the IgG-binding Z-domain, a single-chain variable fragment with specificity for CD38 (scFv