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

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.