Wang Ziyan, Gao Yang, Luo Manyu, Cagliero Cedric, Jiang Hua, Xie Yueqing, Zhu Jianwei, Lu Huili
Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China.
Jecho Laboratories, Inc., 7320 Executive Way, Frederick, MD 21704, USA.
Bio Protoc. 2019 Sep 20;9(18):e3370. doi: 10.21769/BioProtoc.3370.
In comparison with full-length IgGs, antigen binding fragments (Fabs) are smaller in size and do not require the complexed post-translational modification. Therefore, Fab can be cost-effectively produced using an ) expression system. However, the disulfide-bonds containing exogenous protein, including Fab, tend to form insoluble inclusion bodies in , which has been the bottleneck for exogenous protein expressions using this system. The secretory expression of proteins in periplasm or extracellular medium are promising strategies to prevent the formation of inclusion bodies to improve the efficiency to produce Fabs from . The extracellular expression is of particularly interest since it releases the product into the medium, while periplasmic expression yield is limited to the periplasm space. In addition, the extracellular expression allows for the direct harvesting of proteins from the culture supernatant, sparing the procedures of cell lysis and reducing contamination of host cell protein or DNA. Using anti-VEGF Fab as an example, here we provide a protocol based on the alkaline phosphatase (phoA) promoter and the heat-stable enterotoxin II (STII) leader sequence. Using phosphate starvation to induce the secretory expression, the protocol could be generally used for the efficient production of Fabs.
与全长免疫球蛋白（IgG）相比，抗原结合片段（Fab）尺寸更小，且不需要复杂的翻译后修饰。因此，Fab可以使用大肠杆菌表达系统经济高效地生产。然而，包括Fab在内的含二硫键的外源蛋白在大肠杆菌中倾向于形成不溶性包涵体，这一直是使用该系统进行外源蛋白表达的瓶颈。在周质或细胞外培养基中进行蛋白质的分泌表达是防止形成包涵体、提高从大肠杆菌生产Fab效率的有前景的策略。细胞外表达尤其令人感兴趣，因为它将产物释放到培养基中，而周质表达产量限于周质空间。此外，细胞外表达允许直接从培养上清液中收获蛋白质，省去细胞裂解步骤并减少宿主细胞蛋白或DNA的污染。以抗血管内皮生长因子（VEGF）Fab为例，我们在此提供一种基于碱性磷酸酶（phoA）启动子和热稳定肠毒素II（STII）前导序列的方案。利用磷酸盐饥饿诱导分泌表达，该方案可普遍用于高效生产Fab。
