我们开发了一种在大肠杆菌中产生可溶形式的靶向人上皮生长因子受体 2 (HER2) 的单链片段变量 (scFv) 的方法。通过优化可变重域 (VH) 和可变轻域 (VL) 以及 His 标签的方向，我们鉴定出了具有最高 HER2 结合活性的 HL-His 型抗体。 1 L 培养物中 HL-His 的纯化可产生 40.7 mg，纯度 >99%。检测限确定为 2.9 ng，证明了高产量、纯度和灵敏度。此外，我们成功地用荧光染料缀合的 scFv 标记 HER2 细胞系，与 HER2 细胞系相比，观察到的信号背景比明显更高。这凸显了这些荧光 scFv 作为 HER2 乳腺癌诊断有价值的探针的潜力。值得注意的是，完整的 scFv 生产过程得到了简化，只需要 4-5 天。此外，该产品在冷冻储存后仍保持其活性，允许大规模生产和广泛的实际应用。© 2024 作者。生物技术杂志由 Wiley‐VCH GmbH 出版。

We developed a method to produce a soluble form of a single-chain fragment variable (scFv) targeting human epithelial growth factor receptor 2 (HER2) in Escherichia coli. By optimizing the orientations of the variable heavy (VH) and variable light (VL) domains and the His-tag, we identified the HL-His type antibody with the highest HER2-binding activity. Purification of HL-His yielded 40.7 mg from a 1 L culture, achieving >99% purity. The limit of detection was determined to be 2.9 ng, demonstrating high production yield, purity, and sensitivity. Moreover, we successfully labeled HER2+ cell lines with fluorescent dye-conjugated scFv, resulting in a significantly higher observed signal-to-background ratio, compared to that of HER2- cell lines. This highlights the potential of these fluorescent scFvs as valuable probes for HER2+ breast cancer diagnostics. Notably, the process for the complete scFv production was streamlined and required only 4-5 days. Additionally, the product maintained its activity after freeze storage, allowing for large-scale production and a wide range of practical applications.© 2024 The Author(s). Biotechnology Journal published by Wiley‐VCH GmbH.