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Recombinant collagen and collagen-like products are increasingly replacing animal-sourced collagen that is difficult to produce in safe and standard quality. In this study to produce hydroxylated collagen, a 400 base pair collagen fragment of the bovine COL1A1 gene was co-expressed with prolyl-4-hydroxylase subunit α (P4Hα) and prolyl-4-hydroxylase subunit β(P4Hβ) encoding the P4H enzyme in Komagataella phaffii. For this purpose, each target gene was inserted into the pPICZαA vector and then cloned in E. coli DH5α cells. Subsequently, co-expression vectors were constructed using recombinant vectors isolated from positive clones according to the in vitro multimer ligation method. All recombinant expression and co-expression vectors were transformed into K. phaffii X33 cells by electroporation. The results of reverse transcriptase-polymerase chain reaction (PCR) proved that all target genes were transcribed by recombinant strains. The expression of recombinant proteins was performed for 96 hours by methanol-fed cultivation, and the concentration of the purified proteins from the culture medium was measured by the His-Tag enzyme-linked immunosorbent assay (ELISA) method. The concentrations of rP4Hα and rP4Hβ, and rCol1 proteins expressed individually by recombinant strains were determined to be 10.69 µg/L, 10.74 µg/L, and 8.61 µg/L, respectively, while the concentrations of co-expressed rP4Hα/β and rP4Hα/β/rCol1 proteins were 7.82 µg/L and 5.02 µg/L, respectively. These results showed that the target genes were successfully expressed and co-expressed in the recombinant K. phaffii cell.
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