Recombinant expression and functional characterization of human collagen III fragments

Collagen III (COL3) is a key extracellular matrix protein involved in maintaining skin structure and promoting tissue repair. To overcome the instability and expression challenges of full-length human collagen, this study focused on microbial-derived collagen production to develop functionally equivalent and highly stable collagen products. Functional fragments of 30-60 amino acids were extracted from human COL3 to assess whether they could mimic full-length protein activity and promote skin regeneration. Based on melting temperature and net charge, ten fragments were selected and repeated 16-fold for expression in Escherichia coli. Two were soluble, with titers up to 285 and 215 mg/L. These fragments self-assembled into triple helix structures with an untwisting temperature of 25 °C and subsequently formed nanofibers. However, the resulting hydrogels were mechanically weak. Mechanical strength of biomaterials is a crucial factor in promoting cell adhesion and subsequent tissue regeneration. Sample-308 promoted the highest differentiation level of epithelial-like fibroblast (L929) was selected to prepare rigid hydrogel with chitosan (CS), hyaluronic acid (HA) and sodium hyaluronate (SH) separately. The mechanical strength of the sample-308/SH composite hydrogel showed a 10-fold improvement compared to that of the hydrogel prepared with sample-308 alone. It also significantly enhanced fibroblast (L929) adhesion, migration, and differentiation, outperforming both the single-fragment hydrogel and commercial collagen I. In summary, this study developed microbial-expressed bioactive COL3 fragments. The sample-308/SH composite hydrogel showed superior physical properties and substantially improved cellular activity, offering a new solution to develop biomaterials for wound healing.