Key Laboratory of Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing, China.
College of New Energy and Materials, China University of Petroleum, Beijing, China.
Protein Sci. 2022 Jun;31(6):e4335. doi: 10.1002/pro.4335.
Protein-based biomaterials play a significant role in biomedicine and biocatalysis due to their intrinsic biocompatibility and biodegradability. Topological biomaterials show certain advantages without changing the wild-type sequence of the protein, such as unique biofunctions and exceptional stabilities. However, the tuning for the synthesis and assembly of topological protein materials was limited. In this study, we combined the SpyCatcher/SpyTag (SC/ST) chemistry and proposed a cell-free one-pot transcription-translation-assembly system for flexibly regulating the production of topological protein materials. Dimers, trimers, and multimers of proteins with topological structures were designed. Next, the cell-free system was optimized by adjusting the magnesium ion concentration and the molar ratio of different plasmids to obtain the greatest degree of polymerization. The optimal Mg ion concentration was finally determined to be 15 mM, and their most appropriate plasmid molar ratios (SC:ST) were 7:3 for dimers, trimers, and multimers. Finally, based on the topological structure of the polymer, the function was verified with the fusion of xylanase, and it was found that the xylanase activity of the polymer was three times that of the xylanase monomer. Universally, the cell-free system in this study can be used to synthesize protein materials with different topologies based on various covalent or non-covalent methods, and it is likely to have potential in topological structure exploration and bioapplications.
基于蛋白质的生物材料由于其内在的生物相容性和可生物降解性，在生物医学和生物催化中发挥着重要作用。拓扑生物材料在不改变蛋白质野生型序列的情况下表现出某些优势，例如独特的生物功能和卓越的稳定性。然而，拓扑蛋白质材料的合成和组装的调控受到限制。在这项研究中，我们结合了 SpyCatcher/SpyTag（SC/ST）化学，并提出了一种无细胞一锅转录-翻译-组装系统，用于灵活调节拓扑蛋白质材料的生产。设计了具有拓扑结构的蛋白质二聚体、三聚体和多聚体。接下来，通过调整镁离子浓度和不同质粒的摩尔比来优化无细胞系统，以获得最大的聚合度。最终确定最佳的 Mg 离子浓度为 15mM，其最适宜的质粒摩尔比（SC:ST）分别为二聚体、三聚体和多聚体的 7:3。最后，基于聚合物的拓扑结构，通过融合木聚糖酶验证了其功能，发现聚合物的木聚糖酶活性是木聚糖酶单体的三倍。总的来说，本研究中的无细胞系统可以基于各种共价或非共价方法合成具有不同拓扑结构的蛋白质材料，并且很可能在拓扑结构探索和生物应用方面具有潜力。
