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.
Appl Microbiol Biotechnol. 2019 Apr;103(8):3341-3353. doi: 10.1007/s00253-019-09745-8. Epub 2019 Mar 18.
Antigen-binding fragments (Fabs) are an important part of monoclonal antibody (mAb) therapeutics and can be cost-effectively produced using an Escherichia coli (E. coli) expression system. However, Fabs tend to form undesirable aggregates when expressed in the cytoplasm of E. coli, substantially reducing the yield of correctly folded proteins. To solve this problem, in this study, we used five Fab fragments targeting IGF1R, Her2, VEGF, RANKL, and PD-1 to develop a novel system employing the alkaline phosphatase (phoA) promoter and the heat-stable enterotoxin II (STII) leader sequence to facilitate the efficient expression and extracellular secretion of Fabs. Following phosphate starvation, all five Fab fragments were expressed in BL21(DE3), were largely secreted into the culture medium, and then, were further purified by affinity chromatography specific to the constant region of the light chain. The purified Fab products were evaluated and were found to have high purity, antigen-binding affinity, and in vitro bioactivity. The mechanism experiments revealed that (1) BL21(DE3) had significantly higher productivity than the K-12 strains investigated; (2) the secretion ability of the PhoA promoter was superior to that of the T7 promoter; and (3) signal peptide, STII, showed higher extracellular secretion efficiency than pelB. Our findings strongly suggested that the phoA-STII-facilitated extracellular production platform is highly promising for application in the manufacturing of Fab fragments for both academic and industrial purposes.
抗原结合片段（Fabs）是单克隆抗体（mAb）治疗的重要组成部分，可以使用大肠杆菌（E. coli）表达系统经济有效地生产。然而，Fabs 在大肠杆菌的细胞质中表达时往往会形成不理想的聚集体，从而大大降低了正确折叠蛋白的产量。为了解决这个问题，在本研究中，我们使用了针对 IGF1R、Her2、VEGF、RANKL 和 PD-1 的五种 Fab 片段，开发了一种利用碱性磷酸酶（phoA）启动子和热稳定肠毒素 II（STII）前导序列的新型系统，以促进 Fab 的高效表达和细胞外分泌。在磷酸盐饥饿后，所有五种 Fab 片段均在 BL21（DE3）中表达，大部分分泌到培养基中，然后通过针对轻链恒定区的亲和层析进一步纯化。对纯化的 Fab 产物进行评估，发现其具有高纯度、抗原结合亲和力和体外生物活性。机制实验表明：（1）BL21（DE3）的生产力明显高于所研究的 K-12 菌株；（2）PhoA 启动子的分泌能力优于 T7 启动子；（3）信号肽 STII 比 pelB 具有更高的细胞外分泌效率。我们的研究结果强烈表明，phoA-STII 促进的细胞外生产平台非常有希望用于学术和工业目的的 Fab 片段制造。
