Su Yongqin, Zheng Ke, Cheng Cheng
2011 College, Nanjing Tech University, No. 30 Puzhu South Road, Nanjing, 211816, China.
Biomass Molecular Engineering Center and Department of Materials Science and Engineering, School of Forestry and Landscape Architecture, Anhui Agricultural University, Hefei, Anhui, 230036, China.
BMC Biotechnol. 2025 Jul 1;25(1):56. doi: 10.1186/s12896-025-00987-1.
Spidroins are well-known for their exceptional mechanical properties, which have inspired extensive research and applications across various fields. Large-scale production of spidroin continues to face major challenges due to the complexity involved in inducing host organisms to express full-length spidroins. This process requires advanced techniques and has issues such as plasmid instability and potential misfolding.
In this study, we developed a novel expression system by combining a fusion tag with a self-cleavage intein, enabling the convenient expression of three chimeric spidroins with varying numbers of repetitive units. After optimising expression conditions, NT2RepCT, NT4RepCT and NT6RepCT spidroins were obtained in soluble form, with yields of 266, 135 and 125 mg/L, respectively. All three spidroins exhibited an increased β-sheet content with increased numbers of repetitive units during transition from soluble to dry state. In terms of nanofibril morphologies, the self-assemblies of NT4RepCT and NT6RepCT closely resemble those of native silk proteins.
This study can serve as a reference for preparation of high-performance spider silk materials and soluble expression of proteins, such as collagen, and as a foundation for preparation of other structurally complex polymer materials.
蛛丝蛋白以其卓越的机械性能而闻名，这激发了各个领域的广泛研究和应用。由于诱导宿主生物体表达全长蛛丝蛋白过程的复杂性，蛛丝蛋白的大规模生产仍然面临重大挑战。这个过程需要先进的技术，并且存在质粒不稳定和潜在错误折叠等问题。
在本研究中，我们通过将融合标签与自切割内含肽相结合，开发了一种新型表达系统，能够方便地表达具有不同重复单元数量的三种嵌合蛛丝蛋白。优化表达条件后，以可溶形式获得了NT2RepCT、NT4RepCT和NT6RepCT蛛丝蛋白，产量分别为266、135和125mg/L。在从可溶状态转变为干燥状态的过程中，所有三种蛛丝蛋白的β-折叠含量都随着重复单元数量的增加而增加。在纳米纤维形态方面，NT4RepCT和NT6RepCT的自组装与天然丝蛋白非常相似。
本研究可为高性能蜘蛛丝材料的制备以及胶原蛋白等蛋白质的可溶表达提供参考，并为制备其他结构复杂的聚合物材料奠定基础。
