Superdex s75 26 600 column

The plasmid was transformed into E. coli BL21 DE3 cells and cells were grown in TB-MOPS shake culture at 37 °C in the presence of 50 µg/mL kanamycin until the culture reached an optical density of ~ 0.6. Protein expression was induced with 150 µM IPTG and cells were grown at 18 °C over night. Cells were harvested, resuspended in 50 mL buffer A (500 mM NaCl, 50 mM Hepes pH 8.0, 1 mM DTT, 5 mM imidazol, supplemented with one tablet Roche complete Protease Inhibitor Cocktail) and subsequently lyzed by sonication. Lysate was centrifuged for at 1 h at 47,000 rcf and cleared lysate was loaded on a 5 mL QIAGEN Ni–NTA superflow cartridge using a peristaltic pump. The column was washed with 10 CV Buffer A and bound protein was eluted with 5 CV buffer A supplemented with 250 mM imidazol. Eluted protein was incubated with 1% w/w 3C protease at room temperature for 2 h and was subsequently subjected to size exclusion chromatography utilizing a Superdex S75 26/600 column (GE Healthcare) in 500 mM NaCl and 50 mM Hepes pH 7.5. Protein homogeneity and correct mass were verified by SDS-PAGE and LC–MS analysis. Protein was concentrated using a 10,000 MWCO centrifugal concentrator and was subsequently flash-frozen in liquid nitrogen and stored at − 80 °C.

Expression and purification were carried out according to McCaughey et al. (2014). Briefly, the pET21 plasmid containing PL1 was transformed into BL21 DE3 pLyS cells (Agilent, Edinburgh, UK). PL1 expression was induced at mid‐log phase by supplementing the media with 0.3 mm isopropyl β‐D‐1‐thiogalactopyranoside (IPTG), and the cells were grown at 22 °C for 20 h and harvested by centrifugation. The cells were lysed using an MSE Soniprep 150 (Wolf Laboratories, York, UK) and the cell‐free lysate was applied to a 5 mL HisTrap HP column (GE Healthcare, Amersham, UK), and PL1 was eluted using a 5–500 mm imidazole gradient. The remaining contaminants were removed by gel filtration chromatography on a Superdex S75 26/600 column (GE Healthcare, Amersham, UK). PL1 was concentrated using a centrifugal concentrator (Vivaspin 20, Epsom, UK) with a 5 kDa molecular weight cut‐off and stored at −80 °C.