Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 5650871, Japan.
Graduate School of Technology, Industrial and Social Sciences, Tokushima University, 2-1 Minamijosanjima, Tokushima 7708513, Japan; Manufacturing Technology Association of Biologics, 7-1-49 Minatojima-minami, Kobe, Hyogo 6500047, Japan.
J Biosci Bioeng. 2021 Sep;132(3):302-309. doi: 10.1016/j.jbiosc.2021.04.015. Epub 2021 Jun 9.
Cartilaginous fishes such as sharks have adaptive immune systems based on immunoglobulins similar to those in mammals. During their evolution, cartilaginous fishes individually have acquired their adaptive immune system called immunoglobulin new antigen receptor (IgNARs). IgNARs maintain their functions in the harsh environment of shark serum, which contains a high concentration of urea to prevent water loss in seawater. Therefore, IgNARs have high structural stability, and are expected to be used as next-generation antibodies in applications different from those of conventional IgG antibodies. However, no recombinant expression system for IgNAR, which has a molecular weight of approximately 147 kDa as a dimer and multiple N-glycosylation sites, has yet been constructed. This has stalled research into IgNAR development. Here, we constructed a recombinant expression system for IgNAR using Chinese hamster ovary (CHO) cells, widely used as hosts for IgG antibody production. Using this system, IgNAR was successfully expressed and purified as a human IgG Fc fusion protein and showed antigen-binding ability. After Protein A affinity purification, followed by specific cleavage and removal of the human Fc-region, the final yield of IgNAR was 1.07 mg/L-medium. Moreover, this CHO cell expression system modified IgNAR with various N-glycans, including high-mannose and complex types. This expression system will allow us to analyze the structure, physicochemical properties, and biological functions of IgNAR. This fundamental information will advance the development of IgNARs for industrial and biotechnological applications.
软骨鱼类（如鲨鱼）具有基于免疫球蛋白的适应性免疫系统，与哺乳动物的免疫球蛋白相似。在进化过程中，软骨鱼类各自获得了称为免疫球蛋白新抗原受体（IgNAR）的适应性免疫系统。IgNAR 在鲨鱼血清的恶劣环境中保持其功能，鲨鱼血清中含有高浓度的尿素以防止在海水中失水。因此，IgNAR 具有很高的结构稳定性，有望在不同于传统 IgG 抗体的应用中用作下一代抗体。然而，尚未构建出用于 IgNAR 的重组表达系统，IgNAR 作为二聚体的分子量约为 147 kDa，并且具有多个 N-糖基化位点。这阻碍了对 IgNAR 开发的研究。在这里，我们使用中国仓鼠卵巢（CHO）细胞构建了 IgNAR 的重组表达系统，CHO 细胞广泛用作 IgG 抗体生产的宿主。使用该系统，IgNAR 成功地作为人 IgG Fc 融合蛋白表达和纯化，并表现出抗原结合能力。在 Protein A 亲和纯化后，通过特异性切割和去除人 Fc 区，最终 IgNAR 的产量为 1.07 mg/L-培养基。此外，该 CHO 细胞表达系统用各种 N-聚糖修饰 IgNAR，包括高甘露糖型和复杂型。该表达系统将使我们能够分析 IgNAR 的结构、物理化学性质和生物学功能。这些基础信息将推动 IgNAR 在工业和生物技术应用中的发展。
