Isopropyl β d 1 thiogalactopyranoside (iptg)

Bacteriophage proteins were expressed in E. coli BL21(DE3)plysS strain (Promega). Bacterial clones were propagated in LB broth (37 °C with shaking) with kanamycin and chloramphenicol to reach OD600 = 0.8. Protein expression was performed using 0.05 mmol l⁻¹ IPTG (Roche) as an inductor and followed by overnight incubation at 9 °C. Cells were pelleted and suspended in 50 mmol l⁻¹ Tris/HCl pH = 8.0 lysis buffer containing 0.2 mol l⁻¹ NaCl supplemented with protease inhibitor cocktail tablets (Roche). Cells were sonicated 8 times for 30 seconds with 1 minute breaks. After debris removal via centrifugation (14000 × g for 50 minutes) supernatant was mixed with Ni²⁺-agarose beads and incubated at 37 °C for 1 hour on a rotary shaker. After batch chromatography, the beads were washed using lysis buffer to remove unbound proteins. Bounded proteins were eluted using lysis buffer containing 250 mmol l⁻¹ imidazole. Imidazole was removed from the protein’s solution via dialysis on centrifugal filters containing membrane (Millipore) with a cutoff of 3 kDa. The concentration of protein was determined using the BCA method described by Smith et al.^{42 (link)} and eluted fractions were analyzed by SDS-PAGE using 12.5% gels according to the method of Laemmli et al.⁴³ .