SpyCatcher003-mi3 Nanoparticle Production in Bacillus subtilis

SpyCatcher003-mi3 nanoparticles^{83 (link)} were expressed in B. subtilis to avoid endotoxins associated with production in E. coli. Strain CVD175 (B. subtilis 168 ΔaprE ΔnprE Δvpr Δbpr ΔnprB Δmpr Δepr ΔhtrA ΔwprA spoIIE::kan hag::ery; Ingenza Ltd) was transformed with high-copy plasmid pCVD148 encoding for IPTG-inducible expression and secretion of SpyCatcher003-mi3. The resulting strain CVD326 was cultured in Terrific Broth medium (Merck) supplemented with 10 μg/mL chloramphenicol, 10 μg/mL kanamycin and 1% (v/v) glycerol at 37 °C with 250 rpm agitation. When OD₆₀₀ reached a value of 1.0, SpyCatcher003-mi3 expression was induced for 20 hours by addition of IPTG to a final concentration of 1 mM. The culture supernatant was harvested by centrifugation at 4424 × g for 45 minutes at 4°C. SpyCatcher003-mi3 particles were then precipitated by addition of ammonium sulfate at a final concentration of 20% (w/v) with incubation for 45 min at room temperature on a stirrer plate. Precipitated particles were pelleted by centrifugation at 4424 × g for 45 min at 4 °C. The obtained pellet was resuspended in TBS (20 mM Tris, 150 mM NaCl, pH 8.0) and filtered through a 0.22 μm syringe filter. Glycerol was added to a final concentration of 10% (v/v), and purified nanoparticles were stored at −80°C until use.