Expression and Purification of Recombinant Human Type III Collagen using the N‐terminal Transdermal Peptide in <i>Pichia pastoris</i>

To establish a standardized technical framework for large-scale production of recombinant collagen, we have employed Pichia pastoris GS115 as a host expression system and optimized the fermentation and purification processes for a transdermal peptide-type III collagen fusion protein (transdermal peptide-hCOL3A) at the 15-L bioreactor scale. A recombinant vector encoding the fusion gene was constructed through codon optimization and transformed into the GS115 strain. The seed culture was prepared via shake-flask cultivation in BMGY medium. High-cell-density fed-batch fermentation was conducted in a 15-L fermenter with an inoculum size ranging from 3% to 10%, achieving a final recombinant protein yield of 1.9 g/L. In the downstream purification, optimization of membrane-based concentration parameters and the incorporation of strong cation-exchange chromatography using an SP column enhanced the recovery rate of the target protein to 68%. Gel-filtration analysis demonstrated that transdermal peptide-hCOL3A exists as a trimer, and a CCK-8 assay revealed that it promotes proliferation of HaCaT cells in vitro, indicating biological functionality. This work successfully establishes a robust and scalable collagen production process suitable for pilot-scale manufacturing and provides a solid technical foundation for future industrial-scale translation. © 2025 Wiley Periodicals LLC.