Induced Proximity Approach Enables the Recombinant Production of Polyphosphorylated Silk Proteins with Improved...

Phosphorylation is considered to play a role in many of the functional properties of silk proteins, affecting their solubility, environmental adaptability, adhesion, and biocompatibility. However, investigating these effects has been hampered by the difficulty of isolating phosphorylated proteins from natural sources and the limitations of the current in vitro phosphorylation techniques. Here, we present a novel in vivo phosphorylation strategy for recombinant silk proteins in Escherichia coli, utilizing an engineered SpyCatcher/SpyTag system to induce proximity between the target protein and kinase. This scaffolding approach enhances kinase specificity and minimizes off-target effects, increasing the phosphorylation efficiency while preserving cell viability. We demonstrate the applicability of this system to both dragline and aggregate spider silks. Furthermore, we show that polyphosphorylation enhanced the adhesive properties of silk proteins. This modular and tunable strategy provides a powerful platform for producing polyphosphorylated fibrous proteins, offering broad implications for biomaterial design and functional protein engineering.