Purification of Salmonella Pdu Bacterial Microcompartments

S. enterica Pdu BMCs were expressed and purified from an E. coli host R995+PduST (57 (link)). Briefly, A single colony of R995+PduST was used to inoculate 100 mL of 2× YT supplemented with kanamycin and 0.5% 1,2-propanediol and grown overnight at 37°C for ~16 h. The next day, the culture was pelleted; resuspended in 20 mL of a solution containing 50 mM Tris-HCl (pH 8.0), 200 mM KCl, 5 mM MgCl₂, 5 mM β-mercaptoethanol (βME), 0.5 mM EDTA, 0.5 mg/mL lysozyme, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 50% (vol/vol) B-PER (Thermo); gently rocked at room temperature (RT) for 25 min; and then placed on ice for 5 min. Genomic DNA was fragmented by two 1-s pulses from a sonicator. Insoluble matter was pelleted for 20 min at 13,000 × g at 4°C. BMCs were pelleted at 17,500 × g at 4°C for 40 min. Pelleted BMCs were then resuspended in labeling buffer (20 mM phosphate [pH 7.4], 50 mM KCl, 5 mM MgCl₂) and pelleted again for 20 min. The final BMC pellet was resuspended in 1 mL of labeling buffer and stored at 4°C for <2 weeks. BMCs were quantified via a Bradford assay (58 (link)) against a BSA standard.