EcSpy fusion tag dramatically boosts soluble expression of Taq and HER2 nanobodies in E. coli

Recombinant production of single-domain antibodies, known as nanobodies, in Escherichia coli (E. coli) is a cornerstone of their therapeutic and diagnostic development. However, these compact antibody fragments are often prone to aggregation, leading to the formation of insoluble inclusion bodies and drastically reduced functional yields. This bottleneck severely limits the scalability and cost-effectiveness of nanobody manufacturing, hindering their widespread application. Current methods often struggle to achieve high soluble expression, necessitating complex refolding protocols or alternative, more expensive expression systems. Addressing this challenge is crucial for unlocking the full potential of nanobodies in various biomedical fields.

Researchers designed fusion constructs linking the tandem periplasmic chaperone Spheroplast Protein Y from E. coli (EcSpy) with two aggregation-prone nanobodies: Taq-specific and HER2-specific. The EcSpy fusion tag utilized its native signal peptide to direct the nanobodies to the periplasmic space, leveraging its intrinsic chaperone properties to aid proper protein folding. The constructs were expressed in E. coli cultures. Soluble expression was initially assessed via SDS-PAGE during purification steps. After multi-step purification, the yield of both nanobodies was quantified. Proper folding was confirmed using circular dichroism and one-dimensional 1H NMR spectroscopy, while functionality was validated through target-specific immunoassays.

The designed EcSpy fusion strategy markedly enhanced soluble nanobody production, overcoming the typical challenge of inclusion body formation. Quantitative analysis after multi-step purification revealed substantial yields: > From 1 L of E. coli culture, approximately 10.0 mg of HER2 nanobody and 4.0 mg of Taq nanobody were successfully purified. This represents a significant improvement in soluble yield compared to traditional methods for these challenging proteins. Furthermore, the purified nanobodies demonstrated proper structural integrity, as confirmed by circular dichroism and 1H NMR spectroscopy, indicating correct folding. Crucially, both the Taq and HER2 nanobodies retained their specific binding functionality in target-specific immunoassays, confirming that the enhanced solubility did not compromise their biological activity. These findings collectively validate EcSpy as an effective fusion tag for high-yield, soluble, and functional expression of difficult-to-produce nanobodies.