Single‐chain variable fragment (scFv) antibodies have great potential for a range of applications including as diagnostic and therapeutic agents. However, production of scFvs is challenging because proper folding and activity depend on the formation of two intrachain disulfide bonds that do not readily form in the cytoplasm of living cells. Functional expression in bacteria therefore involves targeting to the more oxidizing periplasm, but yields in this compartment can be limiting due to secretion bottlenecks and the relatively small volume compared to the cytoplasm. In the present study, we evaluated an anti‐HER2 scFv, which is specific for human epidermal growth receptor 2 (HER2) overexpressed in breast cancer, for functional expression in the cytoplasm of Escherichia coli strains BL21(DE3) and SHuffle T7 Express, the latter of which is genetically engineered for cytoplasmic disulfide bond formation. Specifically, we observed much greater solubility and binding activity with SHuffle T7 Express cells, which likely resulted from the more oxidative cytoplasm in this strain background. We also found that SHuffle T7 Express cells were capable of supporting high‐level soluble production of anti‐HER2 scFvs with intact disulfide bonds independent of variable domain orientation, providing further evidence that SHuffle T7 Express is a promising host for laboratory and preparative expression of functional scFv antibodies.

中文翻译：

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可变结构域方向对在大肠杆菌细胞质中表达的抗 HER2 单链可变片段抗体的影响

单链可变片段 (scFv) 抗体在包括诊断和治疗剂在内的一系列应用中具有巨大潜力。然而，scFv 的生产具有挑战性，因为正确的折叠和活性取决于两个链内二硫键的形成，而这两个二硫键在活细胞的细胞质中不易形成。因此，细菌中的功能表达涉及靶向更具氧化性的周质，但由于分泌瓶颈和与细胞质相比体积相对较小，该隔室的产量可能会受到限制。在本研究中，我们评估了一种抗 HER2 scFv，它对在乳腺癌中过表达的人表皮生长受体 2 (HER2) 具有特异性，在大肠杆菌细胞质中的功能表达菌株 BL21(DE3) 和 SHuffle T7 Express，后者经过基因工程改造，可形成细胞质二硫键。具体而言，我们观察到与 SHuffle T7 Express 细胞的溶解度和结合活性要大得多，这可能是由于该菌株背景中更具氧化性的细胞质所致。我们还发现，SHuffle T7 Express 细胞能够支持抗 HER2 scFv 的高水平可溶性生产，其具有独立于可变结构域方向的完整二硫键，进一步证明 SHuffle T7 Express 是用于实验室和制备功能性表达的有希望的宿主。 scFv 抗体。