Zhang Chuan, Li An, Wang Ruina, Cao Yan, Jiang Han, Ouyang Songying, Ma Xingyuan, Lu Yiming
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China.
School of Pharmacy, Second Military Medical University, Shanghai, 200433, China.
Protein Expr Purif. 2019 Mar;155:21-26. doi: 10.1016/j.pep.2018.11.002. Epub 2018 Nov 8.
The interaction between TNF-α with TNFR1 triggers important signaling pathways inducing diverse cellular phenomena including inflammation, apoptosis, etc., and is involved in the pathogenesis and progression of numerous autoimmune diseases. The extracellular domain (ECD) of TNFR has been successfully used to clinically treat such TNF-associated diseases. However, large-scale production of these biological material via eukaryotic cell expression systems is usually costly owing to the culture medium and complicated growth conditions. This study aimed to extract pure soluble human TNFR1-ECD and investigate its biological activity, using a prokaryotic expression system. Recombinant vector pMCSG7-TNFR1-ECD was constructed via ligation-independent cloning. The His-tag fusion protein was expressed in E. coli and localized in inclusion bodies. Recombinant TNFR1-ECD was refolded and purified via nickel-affinity chromatography, tag cleavage, followed by cation-exchange chromatography or size-exclusion chromatography. A purity of over 95% and a yield of 9.3 mg protein per liter of bacterial culture media was obtained. The purified protein showed significant affinity of 2.15 nM towards human TNF-α and inhibited TNF-α-mediated cytotoxicity in L929 cells, with an ED of 0.10 μg/ml. It formed a self-associated oligomer with a K of 1.15 μM, detected via microscale thermophoresis. We thus established a highly efficient approach to construct, express, and purify the recombinant protein of human TNFR1-ECD from a prokaryotic system. The antagonistic bioactivities in vitro indicate this protein as a prospective molecules for drug research against autoimmune diseases characterized by TNF-α overexpression.
肿瘤坏死因子-α（TNF-α）与肿瘤坏死因子受体1（TNFR1）之间的相互作用触发重要的信号通路，诱导包括炎症、凋亡等多种细胞现象，并参与众多自身免疫性疾病的发病机制和进展。TNFR的细胞外结构域（ECD）已成功用于临床治疗此类TNF相关疾病。然而，通过真核细胞表达系统大规模生产这些生物材料通常成本高昂，这是由于培养基和复杂的生长条件所致。本研究旨在利用原核表达系统提取纯的可溶性人TNFR1-ECD并研究其生物学活性。通过不依赖连接的克隆构建重组载体pMCSG7-TNFR1-ECD。His标签融合蛋白在大肠杆菌中表达并定位于包涵体中。重组TNFR1-ECD通过镍亲和层析、标签切割，然后进行阳离子交换层析或尺寸排阻层析进行复性和纯化。获得了纯度超过95%的产物，每升细菌培养基的蛋白产量为9.3 mg。纯化后的蛋白对人TNF-α表现出2.15 nM的显著亲和力，并在L929细胞中抑制TNF-α介导的细胞毒性，半数有效剂量（ED）为/ml。通过微量热泳测定，它形成了一个解离常数（K）为1.15 μM的自缔合寡聚体。因此，我们建立了一种高效的方法，用于从原核系统构建、表达和纯化人TNFR1-ECD的重组蛋白。体外拮抗生物活性表明该蛋白是一种针对以TNF-α过表达为特征的自身免疫性疾病进行药物研究的潜在分子。 0.10 μg
