Site-Specific Functionalizationof Recombinant SpiderSilk 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.