Secretion Engineering Enables Production of a Structurally Stable and Long‐Continuous Type III Collagen Fragment in Pichia pastoris

ABSTRACT

Recombinant humanized type III collagen has attracted increasing interest for biomedical and tissue engineering applications due to its roles in extracellular matrix remodeling and tissue repair. However, the production of long‐continuous, structurally stable type III collagen fragment in Pichia pastoris is limited by host‐derived proteolysis of the recombinant α1 chain, resulting in fragmentation and loss of structural integrity. In this study, a long‐continuous type III collagen fragment was produced in P. pastoris by proteolytic selection and identified as a continuous 585‐amino acid sequence (N611–P1195) by LC–MS/MS. Secretion efficiency was improved by signal peptide optimization, enabling the development of high‐producing strains (6.22 g/L). rColIII was purified to > 90% purity with low endotoxin levels (< 10 EU/mg) using multimodal chromatography and exhibited enhanced thermal stability. Although CD analysis indicated the absence of a canonical triple‐helix conformation, FITR spectroscopy confirmed preservation of key peptide backbone structures. Biological assays demonstrated favorable cytocompatibility, with rColIII promoting HSF cell viability, adhesion, and migration. This study provides a scalable strategy for producing long, continuous type III collagen fragment with improved stability in P. pastoris , and supports their potential applications in cosmetic, tissue engineering, regenerative medicine, and other biomedical fields.