Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China.
Tianjin Institute of Industrial Biotechnologygrid.458513.e, Chinese Academy of Sciencesgrid.9227.e, Tianjin, China.
Appl Environ Microbiol. 2022 Apr 26;88(8):e0002822. doi: 10.1128/aem.00028-22. Epub 2022 Apr 6.
Biomaterials offer unique properties that make them irreplaceable for next-generation applications. Fibrous proteins, such as various caterpillar silks and especially spider silk, have strength and toughness not found in human-made materials. In early studies, proteins containing long tandem repeats, such as major ampullate spidroin 1 (MaSp1) and flagelliform silk protein (FSLP), were produced using a large DNA template composed of many tandem repeats. The hierarchical DNA assembly of the DNA template is very time-consuming and labor-intensive, which makes the fibrous proteins difficult to study and engineer. In this study, we designed a circularized mRNA (cmRNA) employing the RNA cyclase ribozyme mechanism. cmRNAs encoding spider silk protein MaSp1 and FSLP were designed based on only one unit of the template sequence but provide ribosomes with a circular and infinite translation template for production of long peptides containing tandem repeats. Using this technique, cmRNAs of MaSp1 and FSLP were successfully generated with circularization efficiencies of 8.5% and 36.7%, respectively, which supported the production of recombinant MaSp1 and FSLP larger than 110 and 88 kDa, containing tens of repeat units. Western blot analysis and mass spectrometry confirmed the authenticity of MaSp1 and FSLP, which were produced at titers of 22.1 and 81.5 mg · liter, respectively. Spider silk is a biomaterial with superior properties. However, its heterologous expression template is hard to construct. The cmRNA technique simplifies the construction and expression strategy by proving the ribosome a circular translation template for expression of long peptides containing tandem repeats. This revolutionary technique will allow researchers to easily build, study, and experiment with any fiber proteins with sequences either from natural genes or artificial designs. We expect a significantly accelerated development of fibrous protein-based biomaterials with the cmRNA technique.
生物材料具有独特的性质，使其成为下一代应用中不可或缺的材料。纤维蛋白，如各种毛毛虫丝，尤其是蜘蛛丝，具有人类制造材料所没有的强度和韧性。在早期研究中，使用由许多串联重复组成的大型 DNA 模板来生产含有长串联重复的蛋白质，例如主要壶腹丝蛋白 1（MaSp1）和鞭毛丝蛋白（FSLP）。DNA 模板的分级 DNA 组装非常耗时耗力，这使得纤维蛋白难以研究和工程化。在这项研究中，我们设计了一种使用 RNA 环化酶核酶机制的环状 mRNA（cmRNA）。根据模板序列的一个单位设计了编码蜘蛛丝蛋白 MaSp1 和 FSLP 的 cmRNA，但为核糖体提供了一个圆形的、无限的翻译模板，用于生产含有串联重复的长肽。使用该技术，成功地生成了 MaSp1 和 FSLP 的 cmRNA，其环化效率分别为 8.5%和 36.7%，这支持了大于 110 和 88 kDa 的重组 MaSp1 和 FSLP 的生产，含有数十个重复单元。Western blot 分析和质谱证实了 MaSp1 和 FSLP 的真实性，它们的产量分别为 22.1 和 81.5 mg·liter。蜘蛛丝是一种具有优异性能的生物材料。然而，其异源表达模板难以构建。cmRNA 技术通过证明核糖体是一个用于表达含有串联重复的长肽的环状翻译模板，简化了构建和表达策略。这项革命性技术将使研究人员能够轻松构建、研究和实验任何具有天然基因或人工设计序列的纤维蛋白。我们预计 cmRNA 技术将显著加速基于纤维蛋白的生物材料的开发。
