Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia coli cytoplasm.
Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia coli cytoplasm.
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DOI：
10.1002/btpr.3102
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发表时间：
2021-03
影响因子：
2.9
通讯作者：
Çelik E
中科院分区：
文献类型：
--
作者：
Koçer İ;Cox EC;DeLisa MP;Çelik E
单链可变片段（scFv）抗体在包括作为诊断剂和治疗剂的一系列应用中具有巨大的潜力。然而，scFv的产生是具有挑战性的，因为正确的折叠和活性取决于两个链内二硫键的形成，而这两个链内二硫键不容易在活细胞的细胞质中形成。因此，细菌中的功能表达涉及靶向氧化性更强的周质，但由于分泌瓶颈和与细胞质相比相对较小的体积，该隔室的产量可能受到限制。在本研究中，我们评估了抗HER2单链抗体，这是特异性的人表皮生长受体2（HER2）在乳腺癌中过表达，在细胞质中的功能性表达。大肠杆菌菌株BL21（DE3）和SHuffle T7 Express，后者被遗传工程改造用于细胞质二硫键形成。具体而言，我们观察到SHuffle T7 Express细胞具有更大的溶解度和结合活性，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细胞能够支持高水平可溶性生产具有完整二硫键的抗HER2 scFv，而与可变结构域方向无关，这进一步证明了SHuffle T7 Express是一种有前途的宿主，可用于实验室和制备性表达功能性scFv抗体。
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 E. 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.
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影响因子：
7.3
作者：
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通讯作者：
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