Bacterial collagen-like proteins differ from vertebrate collagens in that they do not contain hydroxyproline, which is seen as a characteristic of the vertebrate collagens, and which provides a significant contribution to the stability of the collagen triple-helix at body temperature. Despite this difference, the bacterial collagens are stable at around body temperature through inclusion of other stabilising sequence elements. Another difference is the lack of aggregation, and certain vertebrate collagen binding domains that can be introduced into the bacterial sequence lack full function when hydroxyproline is absent. In the present study we have demonstrated that a simple method utilising co-translational incorporation during fermentation can be used to incorporate hydroxyproline into the recombinant bacterial collagen. The presence and amount of hydroxyproline incorporation was shown by amino acid analysis and by mass spectrometry. A small increase in thermal stability was observed using circular dichroism spectroscopy.

Statement of Significance

Recombinant bacterial collagens provide a new opportunity for biomedical materials as they are readily produced in large quantity in E. coli. Unlike animal collagens, they are stable without the need for inclusion of a secondary modification system for hydroxyproline incorporation. In animal collagens, however, introduction of hydroxyproline is essential for stability and is also important for functional molecular interactions within the mammalian extracellular matrix. The present study has shown that hydroxyproline can be readily introduced into recombinant S. pyogenes bacterial collagen through direct co-translational incorporation of this modified imino acid during expression using the codons for proline in the introduced gene construct. This hydroxylation further improves the stability of the collagen and is available to enhance any introduced molecular functions.

中文翻译：

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化脓性链球菌细菌胶原中羟脯氨酸的掺入

细菌胶原蛋白样蛋白质与脊椎动物胶原蛋白的不同之处在于，它们不含羟脯氨酸，羟脯氨酸被视为脊椎动物胶原蛋白的特征，并且在人体温度下对胶原蛋白三螺旋的稳定性起着重要作用。尽管存在这种差异，但细菌胶原蛋白通过包含其他稳定序列元素，可在体温左右保持稳定。另一个区别是缺乏聚集，并且当缺少羟脯氨酸时，可以引入细菌序列的某些脊椎动物胶原结合结构域缺乏完整功能。在本研究中，我们证明了在发酵过程中利用共翻译掺入的简单方法可用于将羟脯氨酸掺入重组细菌胶原蛋白中。通过氨基酸分析和质谱显示了羟脯氨酸掺入的存在和量。使用圆二色光谱法观察到热稳定性的少量增加。

重要声明

重组细菌胶原蛋白为生物医学材料提供了新的机会，因为它们很容易在大肠杆菌中大量生产。与动物胶原蛋白不同，它们稳定，无需包含用于引入羟脯氨酸的二级修饰系统。然而，在动物胶原蛋白中，羟脯氨酸的引入对于稳定性是必不可少的，并且对于哺乳动物细胞外基质内的功能性分子相互作用也很重要。本研究表明，羟脯氨酸可以很容易地引入重组化脓性链球菌中。通过在引入的基因构建体中使用脯氨酸的密码子在表达过程中将修饰的亚氨基酸直接共翻译并入细菌胶原蛋白中。该羟基化进一步改善了胶原的稳定性，并且可用于增强任何引入的分子功能。