[P008] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P04-540] Development of magnetosomes displaying phosphatidylserine-binding proteins for rapid and simple exosome recovery

○Haruhiro Ito¹, Kanata Yuasa¹, Ryoto Tomoe¹, Tomoko Yoshino¹ (1. Graduate School of Engineering, Tokyo University of Agriculture and Technology (Japan))

Keywords:

Magnetotactic bacterium,Magnetosome,Exosome,Phosphatidylserine affinity method

[Purpose]
Exosomes are extracellular vesicles that carry bioactive molecules, including DNA, miRNA, and proteins, and have attracted interest as biomarkers for diagnosis and pathology. For exosome-based analyses, it is essential to establish a recovery method that combines high purity, rapid processing, and preservation of vesicle integrity. Tim4 is a phosphatidylserine (PS)-binding protein used for affinity-based exosome isolation. This approach has attracted attention as an alternative to conventional isolation methods because it enables high-purity recovery while preserving vesicle integrity. However, this method still requires recombinant Tim4 production, purification, and chemical immobilization onto artificial magnetic carriers, making preparation laborious. The magnetotactic bacterium Magnetospirillum magneticum AMB-1 biosynthesizes intracellular magnetic nanoparticles called magnetosomes. In our laboratory, we established a magnetosome display system that enables heterologous proteins to be presented on magnetosomes without chemical conjugation. In this study, we aimed to develop Tim4-displaying magnetosomes as a novel platform for simple and rapid exosome recovery.

[Methods]
To construct Tim4-displaying magnetosomes, expression vectors were designed in which either full-length Tim4 containing the transmembrane domain (fullTim4) or the extracellular region of Tim4 alone (exTim4) was fused to the magnetosome membrane protein Mms13. These constructs were introduced into M. magneticum AMB-1 to generate recombinant strains expressing fullTim4 or exTim4 on magnetosomes. Magnetosomes isolated from the wild-type strain and the transformants were designated WT-mag, fullTim4-mag, and exTim4-mag, respectively. Tim4 expression on magnetosomes and exosome capture capability (using exosome concentrations of 2.5 to 20 ng/ml) were evaluated by ELISA.

[Results and Discussion]
FLAG-tag signals were detected only in magnetosomes from the transformants, indicating successful display of Tim4 on the magnetosome surface. Moreover, exTim4-mag exhibited about 3-fold higher luminescence intensity than fullTim4-mag, indicating a higher display level of Tim4. This difference may reflect reduced expression efficiency or increased misfolding associated with the transmembrane domain in fullTim4. In exosome capture assays, both fullTim4-mag and exTim4-mag showed a positive correlation between exosome concentration and luminescence intensity, indicating that Tim4 retained its PS-binding capability even when displayed on the magnetosome membrane. Notably, fullTim4-mag showed a steeper response than exTim4-mag, suggesting that the transmembrane domain of Tim4 may favorably influence molecular orientation or structural stability, thereby enhancing exosome-binding activity.

[Conclusions]
These findings demonstrate that Tim4-displaying magnetosomes enable rapid and simple exosome recovery and highlight magnetotactic bacteria as a bio-based platform for functional nanomaterials.