Recombinant Phage-Borne Endolysin Production

The recombinant phage-borne endolysin was prepared according to the method described previously by Maciejewska et al.^{34 (link)}. Briefly, the coding sequence for Klebsiella phage KP27 endopeptidase was cloned into the commercially available pEXP-5-CT/TOPO® TA expression vector (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer recommendations, and BL21 (DE3) pLysS (Agilent Technologies, Santa Clara, CA, USA) was transformed with the expression construct. The expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG, final concentration of 0.5 mM), and bacteria further culture additional 18 h at 20 °C. The recombinant protein was purified from the filtered supernatant by affinity chromatography using NGC Medium Pressure Chromatography Systems (Bio-Rad, Hercules, CA, USA) combined with 5-ml nickel columns: Bio-Scale Mini Profinity IMAC Cartridges (Bio-Rad, Hercules, CA, USA) and dialyzed against PBS buffer. The concentration of purified recombinant enzyme was then determined fluorimetrically (Qubit® Protein Assay Kit, Molecular Probes, Thermo Fischer Scientific, Waltham, MA, USA).