A designed fusion tag improves soluble expression of nanobody in E. coli

Nanobodies are small, single-domain antibody fragments derived from camelid heavy-chain antibodies that offer high stability, strong binding affinity, and excellent potential for therapeutic and diagnostic applications. Producing aggregation-prone single-domain antibodies (nanobodies) recombinantly in Escherichia coli (E. coli) remains a significant biotechnological challenge, frequently leading to insoluble inclusion bodies and low functional yields. To overcome this, we designed fusion constructs of the tandem periplasmic chaperone Spheroplast Protein Y from E. coli (EcSpy) with aggregation-prone Taq and HER2-specific nanobodies, employing its native signal peptide to direct periplasmic export and its chaperone property to aid nanobody folding. This approach markedly enhanced soluble nanobody production, as evidenced by SDS-PAGE analysis during purification. After multi-step purification, approximately 10.0 mg of HER2 nanobody and 4.0 mg of Taq nanobody were successfully purified from 1 L of E. coli culture. The purified nanobodies exhibited proper folding, confirmed through circular dichroism and one-dimensional ¹H NMR spectroscopy, and retained their functionality in target-specific immunoassays. These results demonstrate a promising strategy for the high-yield, soluble, and functional expression of challenging nanobodies in E. coli.