Expression and purification of SpyCatcher003-mi3

SpyCatcher003-mi3 was expressed in E. coli BL21(DE3) RIPL cells (Agilent) as previously described¹⁸. Heat-shock transformed cells were then plated on LB-Agar plates (50 µg/mL kanamycin) and incubated for 16 h at 37 °C. A single colony was picked and cultured in 10 mL starter LB culture (50 µg/mL kanamycin) for 16 h at 37 °C and shaking at 200 rpm. The preculture was diluted 1:100 into 1 L LB (50 µg/mL kanamycin and 0.8% (w/v) glucose) and cultured at 37 °C, 200 rpm until OD600 ~0.6. Protein expression was induced with isopropyl β-d-1-thiogalactopyranoside (IPTG) (420 µM) and incubated at 22 °C, 200 rpm for a further 16 h. The culture was centrifuged and the pellet was resuspended in 20 mL 25 mM Tris–HCl, 300 mM NaCl, pH 8.5 with 0.1 mg/mL lysozyme, 1 mg/mL cOmplete mini EDTA-free protease inhibitor (Merck) and 1 mM phenylmethanesulfonyl fluoride (PMSF). Cell suspension was incubated at 22 °C for 30 min on a platform shaker and sonicated on ice four times for 60 s at 50% duty-cycle using an Ultrasonic Processor (Cole-Parmer). Cell lysate was clarified at 35,000 × g for 45 min at 4 °C. The supernatant was filtered through 0.45 and 0.22 µm syringe filters (Starlab) and 170 mg ammonium sulfate was added per mL of lysate. SpyCatcher003-mi3 particles were precipitated by incubating the lysate at 4 °C for 1 h while mixing at 100 rpm. Precipitated particles were pelleted by centrifugation at 30,000 × g for 30 min at 4 °C. The collected pellet was resuspended into 8 mL TBS pH 8.5 (25 mM Tris–HCl, 150 mM NaCl). Residual ammonium sulfate was removed by dialysing for 16 h against 500-fold excess of TBS. Dialysed SpyCatcher003-mi3 was concentrated to 4 mg/mL using a Vivaspin 20 100 kDa spin concentrator (Vivaproducts) and centrifuged at 17,000 × g for 30 min at 4 °C to pellet any insoluble material. The supernatant was filtered through a 0.22 µm syringe filter. The purified SpyCatcher003-mi3 was then further purified using size-exclusion chromatography (SEC). In brief, 2.5 mL was loaded into a HiPrep Sephacryl S-400 HR 16-600 SEC column (GE Healthcare) equilibrated with TBS using an ÄKTA Pure 25 system (GE Healthcare). Proteins were separated at 1 mL/min while collecting 1 mL elution factions. The fractions containing the purified particles were identified by SDS–PAGE, pooled, and concentrated using a Vivaspin 20 100 kDa MW cut-off centrifugal concentrator. Endotoxin was removed from the SpyCatcher003-mi3 samples using Triton X-114 phase separation as previously described¹⁸. The concentration of endotoxin-depleted particles was measured using bicinchoninic acid (BCA) assay (Pierce) and particles were stored at −80 °C.