Huang Yufan, Zhou Bixia, Chen Ziyang, Su Yongqin, Cheng Cheng, He Bingfang
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30 Puzhu South Road, Nanjing, 211816, China.
2011 College, Nanjing Tech University, No. 30 Puzhu South Road, Nanjing, 211816, China.
Microb Cell Fact. 2025 May 14;24(1):108. doi: 10.1186/s12934-025-02734-9.
Spider dragline silk exhibits ultrahigh tensile strength and excellent ductility, making it one of the best-performing natural biomaterials. The major ampullate spidroin (MaSp1) has promising applications in the biomedical, chemical, and military industries owing to its good biocompatibility, biodegradability, and low immunogenicity. The generation of recombinant spidroin can significantly facilitate its scaled production but has several challenges, including the high cost of the downstream spidroin solubilization process and the resulting toxicity due to the use of organic solvents. Unlike common MaSp, MaSp short (MaSp1s) from Cyrtophoramoluccensis is a low-molecular-weight spidroin, lacking the typical repetitive sequences and long poly(A) motif. These features enable the heterologous production of soluble spidroin.
In this study, rMaSp1 and its dimer rMaSp1s-2Core were expressed in soluble form by introducing the SUMO fusion tag and the self-shearing peptide intein. To improve the yield of recombinant spidroin using shake-flask fermentation, response surface analysis was used to optimize the conditions. The yields of rMaSp1 and rMaSp1s-2Core were 218.9 and 95.76 mg/L, respectively. Subsequently, fermentation was scaled up in a 5 L fermenter after adding metal ions and other growth factors to the medium. The optimal inoculation amount, induction temperature, loaded liquid, and feeding strategy were explored. Finally, the yields of rMaSp1 and rMaSp1s-2Core reached 1,112.2 and 297.8 mg/L, respectively. Furthermore, the dimerization of rMaSp1 monomers was achieved by introducing disulfide bonds via exogenous cysteine residues in the C-terminal domain. The secondary structure and self-assembly of rMaSp1 were also analyzed.
This study successfully addressed key challenges in recombinant spidroin production by employing fusion tags (SUMO and self-shearing peptide intein) to enable the soluble expression of rMaSp1 and its dimer rMaSp1s-2Core. The secondary structure and self-assembly analyses further contributed to our understanding of recombinant spidroin. These findings enable the large-scale production of spidroin and its potential applications in the biomedical, chemical, and military industries, overcoming previous hurdles related to the solubility and toxicity associated with downstream processing.
蜘蛛拖牵丝具有超高的拉伸强度和出色的延展性，使其成为性能最佳的天然生物材料之一。主要壶腹蛛丝蛋白（MaSp1）因其良好的生物相容性、生物降解性和低免疫原性，在生物医学、化学和军事工业中具有广阔的应用前景。重组蛛丝蛋白的产生可以显著促进其规模化生产，但也面临一些挑战，包括下游蛛丝蛋白溶解过程的高成本以及使用有机溶剂导致的毒性。与普通MaSp不同，来自 Cyrtophoramoluccensis 的短 MaSp（MaSp1s）是一种低分子量的蛛丝蛋白，缺乏典型的重复序列和长聚（A）基序。这些特性使得能够异源生产可溶性蛛丝蛋白。
在本研究中，通过引入 SUMO 融合标签和自剪切肽内含肽，以可溶形式表达了 rMaSp1 及其二聚体 rMaSp1s - 2Core。为了提高摇瓶发酵中重组蛛丝蛋白的产量，采用响应面分析来优化条件。rMaSp1 和 rMaSp1s - 2Core 的产量分别为 218.9 和 95.76 mg/L。随后，在向培养基中添加金属离子和其他生长因子后，在 5 L 发酵罐中进行了放大发酵。探索了最佳接种量、诱导温度、装液量和补料策略。最终，rMaSp1 和 rMaSp1s - 2Core 的产量分别达到 1112.2 和 297.8 mg/L。此外，通过在 C 末端结构域引入外源半胱氨酸残基形成二硫键，实现了 rMaSp1 单体的二聚化。还分析了 rMaSp1 的二级结构和自组装情况。
本研究通过采用融合标签（SUMO 和自剪切肽内含肽）成功解决了重组蛛丝蛋白生产中的关键挑战，实现了 rMaSp1 及其二聚体 rMaSp1s - 2Core 的可溶性表达。二级结构和自组装分析进一步有助于我们对重组蛛丝蛋白的理解。这些发现使得能够大规模生产蛛丝蛋白及其在生物医学、化学和军事工业中的潜在应用，克服了以往与下游加工相关的溶解性和毒性障碍。
