Optimizing prolyl hydroxylation for functional recombinant collagen in Escherichia coli.

Collagen, a key extracellular matrix component, is renowned for its biocompatibility, biodegradability, and bioactivity, finding wide applications in food, medicine, cosmetics, and industry. Recombinant collagen expression in Escherichia coli offers advantages such as shorter production cycles and lower costs compared to extraction from animal tissues, though it is known to lack essential post-translational modifications, such as proline hydroxylation, which are crucial for its stability and biological function. Studies have shown that certain prolyl hydroxylases, including BaP4H, DsP4H, and L593, exhibit relatively high modification efficiency in the E. coli expression system. However, structures and functions of recombinant human type III collagen after modification by three prolyl hydroxylases remain uncertain. In this study, we investigated the percentage of proline hydroxylation, hydroxylation sites, circular dichroism spectra, and biological functions of recombinant human type III collagen modified by various prolyl hydroxylases. The results indicated that the L593 exhibited the highest percentage of proline hydroxylation, and the percentage of proline hydroxylation was closely associated with the formation of the collagen triple helix, while the hydroxylation ratio of prolines is not positively correlated with the stability of the collagen triple helix structure. The biological function results showed that the cell adhesion of recombinant collagen 3-3(BaP4H) and 3-3(L593) was significantly enhanced, which was closely related to the triple helix structure of recombinant human type III collagen. Our study provides valuable insights into the industrial production and biological applications of collagen, enhancing its functional research and scalability.