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

Authors

Xingyu Wang, Ali Raza, Zhiqing Tao, Guan Wang, Xu Zhang, Maili Liu, Lichun He

Published in

Magnetic resonance letters. Volume 6. Issue 4. Pages 200287. Epub May 15, 2026.

Abstract

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.

PMID:
42436722
Bibliographic data and abstract were imported from PubMed on 12 Jul 2026.

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