Preparation and Characterization of Hydroxylated Recombinant Collagen by Incorporating Proline and Hydroxyproline in Proline-Deficient Escherichia coli

• Published in: Bioengineering (Basel) Vol. 11; no. 10; p. 975
• Main Authors: Cheng, Zhimin, Hong, Bin, Li, Yanmei, Wang, Jufang
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 27.09.2024; MDPI
• Subjects: Amino acids; Binding; Biological products; Biological properties; Biomaterials; Biomedical materials; Body temperature; Cell adhesion & migration; Cell culture; Chemical properties; Cloning; co-expression; Collagen; Culture media; E coli; Escherichia coli; Ethylenediaminetetraacetic acid; Genetic engineering; Hydroxylation; hydroxylation rate; Hydroxyproline; incorporation; Melt temperature; Plasmids; Post-translation; Proline; Proteins; recombinant collagen; Regenerative medicine; RNA polymerase; Structural analysis; Thermal stability; Tissue engineering; triple helix; co-expression; incorporation; triple helix; recombinant collagen; hydroxylation rate
• ISSN: 2306-5354; 2306-5354
• DOI: 10.3390/bioengineering11100975

Collagen possesses distinctive chemical properties and biological functions due to its unique triple helix structure. However, recombinant collagen expressed in without post-translational modifications such as hydroxylation lacks full function since hydroxylation is considered to be critical to the stability of the collagen triple-helix at body temperature. Here, a proline-deficient strain was constructed and employed to prepare hydroxylated recombinant collagens by incorporating proline (Pro) and hydroxyproline (Hyp) from the culture medium. By controlling the ratio of Pro to Hyp in the culture medium, collagen with different degrees of hydroxylation (0-88%) can be obtained. When the ratio of Pro and Hyp was adjusted to 12:8 mM, the proline hydroxylation rate of recombinant human collagen (rhCol, 55 kDa) ranged from 40-50%, which was also the degree of natural collagen. After proline hydroxylation, both the thermal stability and cell binding of rhCol were significantly enhanced. Notably, when the hydroxylation rate approached that of native human collagen (40-50%), the improvements were most pronounced. Moreover, the cell binding of rhCol with a hydroxylation rate of 43% increased by 29%, and the melting temperature (Tm) rose by 5 °C compared to the non-hydroxylated rhCol. The system achieved a yield of 1.186 g/L of rhCol by batch-fed in a 7 L fermenter. This innovative technology is expected to drive the development and application of collagen-related biomaterials with significant application value in the fields of tissue engineering, regenerative medicine, and biopharmaceuticals.