Production and Purification of PM-477 Endolysin

Engineered phage endolysin PM-477 was described previously [20 (link)]. PM-477 was expressed and purified as presented previously [20 (link)]. Briefly, PM-477 expression in E. coli BL21 (DE3) was performed in Terrific Broth Medium, with induction with 1.5% α-lactose monohydrate (Carl Roth) at 25 °C for 24 h, with shaking at 250 rpm. Protein purification was performed by affinity chromatography on a nickel–nitrilotriacetic acid (Ni–NTA) HISTrap column. Elution with 50 mM MES (Carl Roth) pH 7, 150 mM NaCl (Carl Roth), 250 mM imidazole (Carl Roth) was followed by size exclusion chromatography and dialysis against MES buffer (50 mM MES (2-morpholinoethanesulfonic acid) pH 5.5, 100 mM NaCl, 8 mM MgSO₄ (Sigma–Aldrich)). Protein concentration was determined at OD 260/280 nm or by using the PierceTM BCA (bicinchoninic acid) protein assay kit (Thermo Fisher Scientific). Purified PM-477 aliquots (0.5 mg/mL) of 450 µL were stored at −80 °C till the moment of use. The His tag used for purification was not cleaved off PM-477 for the scope of this study.