Cui Jinhui, Wu Dan, Sun Qian, Yang Xiuzhu, Wang Dandan, Zhuang Miao, Zhang Yiheng, Gan Mingzhe, Luo Dan
CAS Key Laboratory of Nano-Bio Interface, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou, China.
School of Nano-Tech and Nano-Bionics, University of Science and Technology of China, Hefei, China.
Front Chem. 2020 Feb 18;8:28. doi: 10.3389/fchem.2020.00028. eCollection 2020.
Cell-free protein synthesis (CFPS) has the advantage of rapid expression of proteins and has been widely implemented in synthetic biology and protein engineering. However, the critical problem limiting CFPS industrial application is its relatively high cost, which partly attributes to the overexpense of single-use DNA templates. Hydrogels provide a possible solution because they can preserve and reutilize the DNA templates in CFPS and have great potential in elevating the protein production yield of the CFPS. Here, we presented a low-cost hybrid hydrogel simply prepared with polyethylene glycol diacrylate (PEGDA) and DNA, which is capable of high-efficient and repeated protein synthesis in CFPS. Parameters governing protein production specific to hybrid hydrogels were optimized. Structures and physical properties of the hybrid hydrogel were characterized. Transcription and expression kinetics of solution phase system and gel phased systems were investigated. The results showed that PEGDA/DNA hydrogel can enhance the protein expression of the CFPS system and enable a repeated protein production for tens of times. This PEGDA/DNA hybrid hydrogel can serve as a recyclable gene carrier for either batch or continuous protein expression, and paves a path toward more powerful, scalable protein production and cell-free synthetic biology.
无细胞蛋白质合成（CFPS）具有蛋白质表达迅速的优点，已在合成生物学和蛋白质工程中得到广泛应用。然而，限制CFPS工业应用的关键问题是其成本相对较高，这部分归因于一次性DNA模板的过度消耗。水凝胶提供了一种可能的解决方案，因为它们可以在CFPS中保存和重复利用DNA模板，并且在提高CFPS的蛋白质产量方面具有巨大潜力。在此，我们展示了一种简单地由聚乙二醇二丙烯酸酯（PEGDA）和DNA制备的低成本混合水凝胶，其能够在CFPS中进行高效且重复的蛋白质合成。对混合水凝胶特有的蛋白质生产控制参数进行了优化。对混合水凝胶的结构和物理性质进行了表征。研究了溶液相系统和凝胶相系统的转录和表达动力学。结果表明，PEGDA/DNA水凝胶可以增强CFPS系统的蛋白质表达，并能够进行数十次的重复蛋白质生产。这种PEGDA/DNA混合水凝胶可作为用于批量或连续蛋白质表达的可回收基因载体，并为更强大、可扩展的蛋白质生产和无细胞合成生物学铺平了道路。
