Recombinant Expression, Structural Identification, and Antioxidant Activity Analysis of Human Type III Collagen in Pichia pastoris
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Abstract:
Collagen plays a vital role in the human body and is widely used in food, health products, and medical treatment. Codon optimization of the human type III collagen gene was carried out according to the codon usage bias of Pichia pastoris. The single-tandem, two-tandem, and four-copy two-tandem expression vectors pPIC9K-COL3-S, pPIC9K-COL3-2, and pPIC9K-COL3-4, respectively, were constructed and transformed into P. pastoris GS115 to achieve integrated expression of human type III collagen, thereby obtaining engineered strains of P. pastoris containing single-tandem collagen, two-tandem collagen, and four-copy two-tandem collagen. The pPIC9K-COL3-S and pPIC9K-COL3-2 recombinant strains were mixed and shaken with a 0.5% induction concentration of methanol to stimulate high-density fermentation. SDS-PAGE and western blot analysis demonstrated that the recombinant strains successfully expressed recombinant collagen, where the apparent molecular weight of the single-tandem protein was approximately 26.7 ku, and that of the two-tandem protein was approximately 52.3 ku. The yield of the high-density shake flask fermentation of the four-copy recombinant strain induced by 0.5% methanol was the highest, with an optimal induction time of 72 h and protein yield reaching approximately 0.45 g/L. A high-purity recombinant protein of this strain was obtained after purification using a nickel column. Antioxidant activity experiments showed that the DPPH free-radical scavenging rate of the recombinant collagen reached 51.49%, whereas the ABTS free-radical scavenging rate reached 41.24%, thus proving its antioxidant activity. This provides a theoretical basis for its application in the fields of food, health products, and medicine.
