Tagawa, A. | Search bioRxiv

bioRxiv2024

Development of nanobodies against human Survivin, applicable to suppress cell proliferation.

Survivin, a member of the inhibitor of apoptosis protein (IAP) family, is overexpressed in many cancers, making it a valuable target for therapy and a biomarker for cancer diagnostics. As Survivin contributes to cancer development by inhibiting apoptosis and promoting rapid cell division, inhibiting its activity is a promising strategy to overcome cancer resistance to apoptosis and reduce tumor growth. In this study, we developed nanobodies targeting human Survivin using M13 phage display and deep sequencing to identify high-affinity binders. Seven nanobody clones were selected and evaluated through ELISA, western blotting, and immunostaining. Clones 7 and 15, which specifically bind the alpha-helix region of Survivin, effectively suppressed cell proliferation by disrupting cell cycle regulation, likely by interfering interaction with intracellular binding partners. These findings suggest that Survivin-targeting nanobodies hold potential as therapeutic agents for cancer treatment, offering a novel approach to inhibiting tumor growth through the disruption of Survivin-mediated processes.

cancer biology↗

bioRxiv2024

Efficient production of functional proaerolysin from E.coli

Proaerolysin is a bacterial toxin produced by Aeromonas hydrophila that specifically binds to GPI-anchored proteins on the plasma membrane, creating transmembrane pores that lead to cell death within a few hours. Leveraging this unique property, proaerolysin is widely used in diagnostic tests for paroxysmal nocturnal hemoglobinuria (PNH), a disease caused by somatic mutations in the PIGA gene, which is involved in the biosynthesis of GPI anchors. Additionally, proaerolysin serves as a counter-selection agent in genetic manipulations. Although bacterial expression and purification of proaerolysin have been previously reported, yields were low due to the absence of internal disulfide bonds, which are crucial for protein stability. Here, we demonstrate that using the Shuffle E. coli strain, which facilitates the formation of disulfide bonds in the cytoplasm, significantly improves the solubility and proper folding of proaerolysin. We achieved a high yield of proaerolysin, approximately 3 mg from a 50 ml bacterial culture, with a purity of over 99%. The functionality of recombinant proaerolysin was confirmed by testing in mouse embryonic stem cells (mESCs), demonstrating that this high-yield production method offers a reliable and cost-effective source of functional proaerolysin for a wide range of biotechnological applications.

biochemistry↗