Kim Young Su, Lee Hye-Jeong, Kim Mi-Reu, Jeong Hwabong, Kim Young Pil, Park Jung-Ho, Ahn Jungoh
Biotechnology Process Engineering Center, KRIBB, Cheongju, 20736, Republic of Korea.
Bio-Evaluation Center, KRIBB, Cheongju, 20736, Republic of Korea; Department of Applied Biological Engineering, KRIBB School of Biotechnology, Korea University of Science and Technology (UST), 217 Gajeong-Ro, Yuseong-Gu, Daejeon, Republic of Korea.
Protein Expr Purif. 2025 Nov;235:106786. doi: 10.1016/j.pep.2025.106786. Epub 2025 Jul 30.
Human plasma gelsolin (pGSN) is an 83 kDa actin-binding protein involved in cytoskeletal remodeling, inflammation, and host defense. Its clinical relevance as a biomarker and potential therapeutic agent, particularly in conditions like sepsis, acute respiratory distress syndrome (ARDS), and cystic fibrosis, has driven interest in scalable recombinant expression. However, high-yield production of functionally active gelsolin is hindered by its complex structure and folding requirements. To address this, we developed a scalable, high-yield bacterial expression system that achieves among the highest reported levels of functional recombinant human gelsolin (rGelsolin) using a GST-fusion strategy incorporating a tobacco etch virus (TEV) protease cleavage site, optimized for solubility and downstream processing. High-density fed-batch fermentation in E. coli yielded 5.0 g/L of soluble protein. Following a three-step purification process with removal of the GST tag, 2.1 g/L of tag-free, high-purity rGelsolin with >95 % purity was obtained. Structural characterization by circular dichroism spectroscopy confirmed that rGelsolin adopted a native-like secondary structure and exhibited thermal stability (Tm ∼59 °C). Correct processing of the recombinant protein was verified by N- and C-terminal sequencing. Functional assays demonstrated that rGelsolin bound to and severed actin filaments in a calcium-dependent manner, similar to native plasma gelsolin. These findings demonstrate a scalable, cost-effective platform for producing bioactive rGelsolin in E. coli, with structural and functional features comparable to native pGSN, supporting its potential utility in diagnostic, therapeutic, and structural applications in the context of acute and chronic inflammatory diseases.
人血浆凝溶胶蛋白（pGSN）是一种83 kDa的肌动蛋白结合蛋白，参与细胞骨架重塑、炎症反应和宿主防御。其作为生物标志物和潜在治疗剂的临床相关性，特别是在脓毒症、急性呼吸窘迫综合征（ARDS）和囊性纤维化等病症中，引发了人们对可扩展重组表达的兴趣。然而，功能性活性凝溶胶蛋白的高产生产受到其复杂结构和折叠要求的阻碍。为了解决这一问题，我们开发了一种可扩展的高产细菌表达系统，该系统采用包含烟草蚀纹病毒（TEV）蛋白酶切割位点的GST融合策略，实现了功能重组人凝溶胶蛋白（rGelsolin）的高水平表达，该策略针对溶解性和下游加工进行了优化。在大肠杆菌中进行高密度补料分批发酵，可产生5.0 g/L的可溶性蛋白。经过三步纯化过程去除GST标签后，获得了2.1 g/L的无标签、高纯度rGelsolin，纯度>95%。通过圆二色光谱进行的结构表征证实，rGelsolin采用了类似天然的二级结构并表现出热稳定性（Tm ∼59 °C）。通过N端和C端测序验证了重组蛋白的正确加工。功能测定表明，rGelsolin以钙依赖的方式结合并切断肌动蛋白丝，类似于天然血浆凝溶胶蛋白。这些发现证明了一种在大肠杆菌中生产生物活性rGelsolin的可扩展、经济高效的平台，其结构和功能特征与天然pGSN相当，支持其在急性和慢性炎症疾病的诊断、治疗和结构应用中的潜在效用。
