Nanobody fusion enhances production of difficult-to-produce secretory proteins

Secretory protein expression in mammalian cells is widely used in various fields, including biomedical research and biopharmaceutical production. However, achieving high-level expression of certain secretory proteins/peptides can be challenging. The naturally occurring N1 fragment of the prion protein is one of these difficult-to-produce secretory proteins, which hinders our understanding of its biological functions and limits its potential as a therapeutic molecule. To improve N1 production, we screened several well-folded protein domains and found that fusing N1 with a camelid nanobody (Nb) improved its translocation into the endoplasmic reticulum and significantly enhanced its secretion. Nb fusion does not alter the translocation mechanism, which remains dependent on the Sec61-Sec62-Sec63 complex. This approach also resulted in a significant increase in N1 production in the mouse brain using recombinant adeno-associated virus. Furthermore, fusing Nb to another unstructured protein, Shadoo (without glycosylphosphatidylinositol anchor), or a peptide hormone, somatostatin, also greatly increased their production, demonstrating the applicability of this approach to other proteins and peptides. The enhancement of N1 production is comparable or better than Fc fusion, and the effect is observed with all tested camelid Nb but not with a shark Nb and to a lesser extent with a human immunoglobulin heavy chain variable region. Importantly, the Nb in the fusion protein retained its antigen-binding capability, paving the way for the development of a dual-functional protein. Collectively, we present a novel strategy for enhancing the production of secretory proteins, which holds great promise in creating functional biological molecules for a wide range of applications.