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

## TL;DR

Scientists engineered silkworms to produce a human protein that supports nerve growth, which could help in treating nerve injuries and diseases.

## Contribution

A transgenic silkworm system for scalable production of bioactive human NT-3 protein using the silk gland bioreactor.

## Key findings

- NT-3 was successfully expressed in silkworm silk glands and secreted into silk fibers at 0.5 mg per gram of cocoon weight.
- NT-3-functionalized silk enhanced neural cell proliferation, migration, differentiation, and neurite outgrowth without cytotoxicity.
- The recombinant NT-3 retains high bioactivity, showing promise for nerve regeneration and tissue engineering applications.

## Abstract

The silkworm, Bombyx mori, a commercially vital resource for sericulture, produces natural silk with extensive applications spanning textiles, biomedical engineering, and advanced material science. By using the silkworm middle silk gland-specific expression system, we developed a transgenic silkworm strain capable of synthesizing human neurotrophic factor 3 (NT-3) protein, a critical neurotrophic factor essential for neural development, cellular survival, and synaptic plasticity. The resulting NT-3-enriched sericin matrix demonstrated enhanced biological functionality, significantly promoting cell proliferation, migration, differentiation, and neurite outgrowth in mouse hippocampal HT-22 neuron cells. These findings not only establish B. mori as an efficient bioreactor for large-scale NT-3 production but also reveal a dual function of NT-3 for the preservation of bioactivity and programmable release kinetics, which makes it a promising candidate for treating peripheral neuropathies and spinal cord injuries.

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.