Microfluidize m 110p

Strain BL21(DE3) Rosetta pLysS Rare (Cm^R) of E. coli was transformed with the plasmid pET28-His-LytA (Kan^R) or pET28-His-LytAE₈₇Q (Kan^R). Cells were grown in Luria Bertani medium and protein expression was induced at OD_595nm 0.6 with 0.5 mM IPTG (isopropyl β-D-thiogalactopyranoside) at 25°C overnight. Cells from 2-litres cultures were harvested by centrifugation, resuspended in 50 ml of a buffer containing 50 mM Tris pH 8.0, 500 mM NaCl, 25 mM imidazole, 10% glycerol and a protease inhibitor cocktail (Complete EDTA free, Sigma-Aldrich) and lyzed using a Microfluidize M-110P (Microfluidics). The lysate was clarified by centrifugation (20 min at 39,191 × g at 4°C) and loaded onto a 10-ml Ni-nitrilotriacetic acid (NTA) column (Qiagen). His-LytA proteins were eluted with a 25 mM to 500 mM imidazole gradient. Pooled fractions were concentrated and further purified by size exclusion chromatography using a Superdex S75 10/300 GL column (GE Healthcare) in 25 mM Tris-HCl (pH 8) and 150 mM NaCl.