[Preparation and characterization of recombinant human-source collagen]

Area of Science:

• Biotechnology
• Biomaterials Science
• Molecular Biology

Background:

• Collagen is a crucial structural protein in the human body, essential for tissue integrity and function.
• The industrial production of human-source collagen presents challenges, necessitating the development of recombinant methods.
• Type I collagen, the most abundant collagen type, is particularly relevant for biomedical applications.

Purpose of the Study:

• To develop and industrialize a recombinant human-source collagen.
• To optimize the production process for high yield and purity.
• To characterize the structural and functional properties of the produced collagen.

Main Methods:

• Design of human collagen amino acid and nucleotide sequences based on the Gly-X-Y motif.
• Construction of the pPIC9K-COL expression vector.
• Electrotransformation of Pichia pastoris to create an engineering strain.
• Fermentation, purification, and identification of recombinant human collagen.
• Characterization using N-terminal sequencing, molecular weight analysis, amino acid analysis, collagenase degradation, scanning electron microscopy, and cytotoxicity assays.

Main Results:

• Achieved a high expression level of 4.5 g/L with over 95% purity.
• Confirmed the primary structure consistency with the designed human-source collagen.
• Demonstrated a porous fiber reticular structure via scanning electron microscopy.
• Showcased superior cytocompatibility through cell cytotoxicity tests.

Conclusions:

• Successfully produced recombinant human-source collagen with high yield and purity.
• The produced collagen possesses desirable structural and cytocompatibility properties for biomedical use.
• This work provides a foundation for the further industrial application of human-source collagen as a biomedical material.