The overexpression of human epidermal growth factor receptor 2 (HER2) is recognized as a hallmark of several solid tumors, and the anti-HER2 monoclonal antibody and its engineered variants have been developed as therapeutic or diagnostic tools for HER2-overexpressing cancers. Here, we demonstrate the first example of extracellular production of an anti-HER2 single-chain variable fragment (scFv) in Escherichia coli via a non-peptide-guided secretion. The initial construct expressing an engineered green fluorescent protein-fused scFv was successful in extracellular secretion, but the expressed protein appeared to be readily truncated before the final purification. Alternatively, by adding Triton X-100 to the culture media, maltose-binding protein (MBP)-fused scFv could be secreted without significant truncation, and homogeneous purification of the intact protein was enabled. The affinity of the MBP-fused scFv to the extracellular domain of HER2 was confirmed by enzyme-linked immunosorbent assay, and the dissociation constant of the finally isolated scFv estimated by surface plasmon resonance was approximately 0.2 nM, which was superior to the other known anti-HER2 scFvs. Given that anti-HER2 scFv is an invaluable asset for developing various therapeutic or diagnostic agents for cancers, we expect that our method for the extracellular production will enable a better manufacturing process for its industrial application.

人表皮生长因子受体 2 (HER2) 的过度表达被认为是几种实体瘤的标志，抗 HER2 单克隆抗体及其工程变体已被开发为 HER2 过度表达癌症的治疗或诊断工具。在这里，我们展示了在大肠杆菌中通过细胞外生产抗 HER2 单链可变片段 (scFv) 的第一个例子一种非肽引导的分泌物。表达工程化绿色荧光蛋白融合 scFv 的初始构建体在细胞外分泌中取得成功，但表达的蛋白质在最终纯化之前似乎很容易被截断。或者，通过将 Triton X-100 添加到培养基中，麦芽糖结合蛋白 (MBP) 融合的 scFv 可以在没有明显截断的情况下分泌，并且能够对完整蛋白质进行均质纯化。MBP融合的scFv对HER2胞外域的亲和性通过酶联免疫吸附试验证实，最终分离的scFv通过表面等离子体共振估计的解离常数约为0.2 nM，优于其他已知的抗-HER2 scFvs。