LAUSR

Abstract 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.