Mizukami Makoto, Onishi Hiromasa, Hanagata Hiroshi, Miyauchi Akira, Ito Yuji, Tokunaga Hiroko, Ishibashi Matsujiro, Arakawa Tsutomu, Tokunaga Masao
R & D Department, Higeta Shoyu Co., Ltd, 2-8 Chuo-cho, Choshi, Chiba, 288-8680, Japan.
Graduate School of Science and Engineering, Kagoshima University, 1-21-35 Korimoto, Kagoshima, 890-0065, Japan.
Protein Expr Purif. 2018 Oct;150:109-118. doi: 10.1016/j.pep.2018.05.013. Epub 2018 May 29.
The Brevibacillus expression system has been successfully employed for the efficient productions of a variety of recombinant proteins, including enzymes, cytokines, antigens and antibody fragments. Here, we succeeded in secretory expression of Trastuzumab Fab antibody fragments using B. choshinensis/BIC (Brevibacillus in vivocloning) expression system. In the fed-batch high-density cell culture, recombinant Trastuzumab Fab with amino-terminal His-tag (His-BcFab) was secreted at high level, 1.25 g/liter, and Fab without His-tag (BcFab) at ∼145 mg/L of culture supernatant. His-BcFab and BcFab were purified to homogeneity using combination of conventional column chromatographies with a yield of 10-13%. This BcFab preparation exhibited native structure and functions evaluated by enzyme-linked immunosorbent assay, surface plasmon resonance, circular dichroism measurements and size exclusion chromatography. To our knowledge, this is the highest production of Fab antibody fragments in gram-positive bacterial expression/secretion systems.
短短芽孢杆菌表达系统已成功用于高效生产多种重组蛋白，包括酶、细胞因子、抗原和抗体片段。在此，我们利用嗜碱短短芽孢杆菌/体内克隆（BIC）表达系统成功实现了曲妥珠单抗Fab抗体片段的分泌表达。在补料分批高密度细胞培养中，氨基末端带有His标签的重组曲妥珠单抗Fab（His-BcFab）以1.25 g/升的高水平分泌，而无His标签的Fab（BcFab）在培养上清液中的浓度约为145 mg/L。通过传统柱色谱组合将His-BcFab和BcFab纯化至均一，产率为10-13%。通过酶联免疫吸附测定、表面等离子体共振、圆二色性测量和尺寸排阻色谱评估，该BcFab制剂具有天然结构和功能。据我们所知，这是革兰氏阳性细菌表达/分泌系统中Fab抗体片段的最高产量。
