Site-Specific Functionalization of Recombinant Spider Silk Using Enzymatic Sortase Coupling

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_CD38), and the antibacterial endolysin Sal-1. The target proteins were produced with an LPETGG sortase recognition tag at the C-terminus to enable coupling. Moreover, a comparative analysis of sortase coupling efficiency of the target proteins was performed using two different silk protein variants, FN-4RepCT with one N-terminal glycine (G-silk) and five N-terminal glycines (G₅-silk). The functionalized silks were assessed by using protein gel electrophoresis, fluorescence microscopy, surface plasmon resonance, and a biochemical assay. Results showed that G₅-silk is more efficient for sortase coupling of the target proteins in solution as well as to silk coatings, when compared to G-silk. In all cases, the target proteins, the Z-domain, the scFv_CD38 fragment, and Sal-1, retained their specific activity after sortase coupling. To conclude, the sortase coupling strategy is a mild and efficient approach to functionalize various silk formats with small (Z-domain) or larger (scFv_CD38, Sal-1) functional molecules.