Chen Quan, Zhou Yuhang, Yu Jianli, Liu Wenshuai, Li Fei, Xian Mo, Nian Rui, Song Haipeng, Feng Dongxiao
CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, 266101, China.
Shenzhen Innova Nanobodi Co., Ltd., No. 7018 Caitian Road, Shenzhen, 518000, China.
Protein Expr Purif. 2019 Mar;155:43-47. doi: 10.1016/j.pep.2018.11.001. Epub 2018 Nov 8.
Nanobodies offer multiple advantages over conventional antibodies in terms of size, stability, solubility, immunogenicity, and production costs, with improved tumor uptake and blood clearance. Additionally, the recombinant expression of nanobodies is robust in various expression systems, such as Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris. P. pastoris is the most widely used microorganism for nanobody production, but all or almost all expression vectors developed for this system are based on the regulated promoter of the alcohol oxidase 1 gene (AOX1) that requires methanol for full induction. In this study, a constitutive anti-CEACAM5 nanobody expression system was constructed under the control of a glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) promoter. The effects of different carbon sources and pH on nanobody expression were evaluated in shaking flask cultures. After 96 h of constitutive expression in shaking flask, a yield of 51.71 mg/L was obtained. In addition, this constitutive expression system produced nanobodies at equivalent yield and affinity to that produced by methanol-induced expression. The results of this study indicated that the use of a constitutive expression system is a promising alternative for the production of nanobodies applied for cancer diagnosis and therapy.
在大小、稳定性、溶解性、免疫原性和生产成本方面，纳米抗体比传统抗体具有多种优势，且肿瘤摄取和血液清除得到改善。此外，纳米抗体在各种表达系统中，如大肠杆菌、酿酒酵母和巴斯德毕赤酵母中，重组表达都很稳定。巴斯德毕赤酵母是用于生产纳米抗体最广泛使用的微生物，但为该系统开发的所有或几乎所有表达载体都基于需要甲醇进行完全诱导的醇氧化酶1基因（AOX1）的调控启动子。在本研究中，构建了一种在甘油醛-3-磷酸脱氢酶启动子（GAP）控制下的组成型抗癌胚抗原相关细胞黏附分子5纳米抗体表达系统。在摇瓶培养中评估了不同碳源和pH对纳米抗体表达的影响。在摇瓶中进行96小时的组成型表达后，产量达到51.71 mg/L。此外，该组成型表达系统产生的纳米抗体产量和亲和力与甲醇诱导表达产生的纳米抗体相当。本研究结果表明，使用组成型表达系统是生产用于癌症诊断和治疗的纳米抗体的一种有前景的替代方法。
