Enhancement of a Humanized Nanobody against HER2 Tyrosine Kinase Production in the Cytoplasm of Escherichia
coli

Around 30% of invasive breast cancers are associated with the overexpression of the human epidermal growth factor receptor 2 (HER2) protein. While monoclonal antibodies are commonly used in targeted therapies, their large size hinders efficient delivery to tumor cells. Nanobodies, singledomain antibody fragments, provide a promising alternative due to their high binding affinity and small size. VHH17, a nanobody specific to HER2-Tyrosine Kinase (HER2-TK), blocks phosphorylation and reduces cancer cell proliferation. This study aimed to develop a humanized anti-HER2-TK nanobody and optimize its production in the cytoplasmic space of E. coli. The effects of temperature and induction on soluble expression were evaluated. Western blot analysis revealed that 20 °C and 1 mM IPTG induction were optimal for humanized VHH17 expression. These results demonstrate that E. coli can effectively express humanized VHH17 under proper conditions. Further optimization may enhance the yield of soluble VHH production.