CSIRO Manufacturing, Bayview Avenue, Clayton, VIC 3169, Australia.
CSIRO Manufacturing, Bayview Avenue, Clayton, VIC 3169, Australia.
Acta Biomater. 2018 Oct 15;80:169-175. doi: 10.1016/j.actbio.2018.09.012. Epub 2018 Sep 13.
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.
细菌胶原蛋白与脊椎动物胶原蛋白不同，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之一，对胶原蛋白在体温下的三螺旋结构的稳定性有重要贡献。尽管存在这种差异，但细菌胶原蛋白通过包含其他稳定的序列元件，在体温左右仍保持稳定。另一个区别是缺乏聚集，并且某些可以引入细菌序列的脊椎动物胶原蛋白结合结构域在不存在羟脯氨酸时缺乏完整的功能。在本研究中，我们已经证明，一种利用发酵过程中共翻译掺入的简单方法可以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法证明了羟脯氨酸掺入的存在和数量。圆二色性光谱分析观察到热稳定性略有提高。意义声明：重组细菌胶原蛋白为生物医学材料提供了新的机会，因为它们可以在大肠杆菌中大量生产。与动物胶原蛋白不同，它们不需要包含羟脯氨酸掺入的二级修饰系统即可稳定。然而，在动物胶原蛋白中，引入羟脯氨酸对于稳定性至关重要，对于哺乳动物细胞外基质中的功能分子相互作用也很重要。本研究表明，通过在表达过程中直接共翻译掺入这种修饰的亚氨基酸，可以将羟脯氨酸轻易地引入重组 S. pyogenes 细菌胶原蛋白中，该亚氨基酸是在引入的基因构建体中用脯氨酸的密码子引入的。这种羟化作用进一步提高了胶原蛋白的稳定性，并可用于增强任何引入的分子功能。
