Efficient expression of full-length triple-helix recombinant type III collagen in Pichia pastoris

Type III collagen (COL3A1) is a significant fibrillar collagen abundant in vascular and cutaneous tissues, playing vital roles in wound healing and tissue repair. The growing demand for COL3A1 in biomedical and cosmetic applications has driven the development of recombinant production systems. In this study, we established a Pichia pastoris platform for the secretory expression of full-length human COL3A1. First, we tested and compared two strategies to promote the formation of a triple-helix structure: co-expression of prolyl 4-hydroxylase (P4H) and fusion with the C-terminal trimerization domain of T4-fibritin. Structural analyses revealed that the T4-fibritin fusion was markedly more effective than P4H co-expression in driving triple-helix formation and enabling subsequent fibrillar self-assembly. Functionally, the T4-fibritin-fused COL3A1 exhibited optimal bioactivity, significantly promoting the proliferation and migration of mesenchymal stem cells as well as accelerating blood coagulation in vitro. Furthermore, the expression system was optimized by screening signal peptides and promoters and by adjusting fermentation parameters, which increased the yield of full-length human COL3A1 to 0.22 g/L in 250 mL shake flasks. Finally, scale-up was carried out in a 5-L bioreactor, and the medium composition was optimized to inhibit protein degradation, resulting in a yield of 0.9 g/L. This study lays the foundation for the industrial production of bioactive, full-length triple-helix human COL3A1, thereby paving the way for its application as a functional biomaterial in skin repair and regenerative medicine.