Cheng Zhimin, Hong Bin, Li Yanmei, Wang Jufang
School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China.
Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China University of Technology, Guangzhou 510006, China.
Bioengineering (Basel). 2024 Sep 27;11(10):975. 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.
由于其独特的三螺旋结构，胶原蛋白具有独特的化学性质和生物学功能。然而，在没有诸如羟基化等翻译后修饰的情况下表达的重组胶原蛋白缺乏完整功能，因为羟基化被认为对胶原蛋白三螺旋在体温下的稳定性至关重要。在此，构建了一种脯氨酸缺陷型菌株，并通过从培养基中掺入脯氨酸（Pro）和羟脯氨酸（Hyp）来制备羟基化重组胶原蛋白。通过控制培养基中Pro与Hyp的比例，可以获得不同羟基化程度（0 - 88%）的胶原蛋白。当Pro和Hyp的比例调整为12:8 mM时，重组人胶原蛋白（rhCol，55 kDa）的脯氨酸羟基化率在40 - 50%之间，这也是天然胶原蛋白的程度。脯氨酸羟基化后，rhCol的热稳定性和细胞结合能力均显著增强。值得注意的是，当羟基化率接近天然人胶原蛋白的羟基化率（40 - 50%）时，改善最为明显。此外，与未羟基化的rhCol相比，羟基化率为43%的rhCol的细胞结合能力提高了29%，解链温度（Tm）升高了5°C。该系统通过在7 L发酵罐中分批补料实现了rhCol的产量为1.186 g/L。这项创新技术有望推动胶原蛋白相关生物材料的开发和应用，在组织工程、再生医学和生物制药领域具有重要的应用价值。
