Periplasmic Protein Expression in E. coli

Expression was performed in E. coli BL 21 codonplus using a pET-22b vector system with a pelB-leader sequence for export to the periplasm of E.coli. After transformation cells were cultivated at 180 rpm and 30°C in shaking flasks in 400 mL LB-medium. After the OD600 had reached 0.4, expression was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG, Carl Roth, final concentration 0.2 mM). The expression proceeded over 15 h at 20°C. The cells were harvested by centrifugation at 4700 rpm for 20 min at 4°C (Heraeus Multifuge X3 FR). Protein purification from the periplasm was carried out by osmotic shock in ddH₂O, using the protocol by Petersen et al. [53 (link)]. The supernatant was frozen in liquid nitrogen and lyophilized overnight (Lyophilizer, Beta 1–8 Martin Christ). The product was analyzed by SDS-polyacrylamide-gel electrophoresis (SDS-PAGE, S2 Fig).