Biosynthesis of Bioactive Human Neurotrophic Factor 3 in Silkworms and Its Biomedical Applications

Neurotrophic factor 3 (NT-3), a potent neurotrophin, promotes neuronal survival and axonal regeneration while demonstrating a unique capacity to induce lineage-specific differentiation of pluripotent stem cells into functional neurons, underscoring its therapeutic potential in neural repair. Despite these advantages, the large-scale production of recombinant human NT-3 with preserved structure integrity and functional bioactivity remains a critical challenge. This study takes advantage of the silk gland bioreactor of silkworms for the recombinant expression of human NT-3 protein on a large scale. Our findings reveal that NT-3 was successfully expressed in the middle silk gland of silkworms and secreted into the silk fibers, achieving a yield of up to 0.5 mg of bioactive NT-3 per gram of cocoon weight. The engineered NT-3-functionalized silk material demonstrates no cytotoxicity and significantly enhanced the proliferation, migration, and differentiation of neural cells compared to natural silk protein. Importantly, this functionalized material also promotes neurite outgrowth in HT-22 cells. These results collectively underscore the high bioactivity of the recombinant human NT-3 protein produced in the silkworm silk gland. The ongoing fabrication of NT-3-incorporated silk-based materials holds considerable promise for advancing tissue engineering and nerve regeneration applications.