Isolation of a Novel Lytic Bacteriophage against a Nosocomial Methicillin-Resistant Staphylococcus aureus Belonging to ST45

Figure 4

Visualization of the expressed recombinant R4lys endolysin. Comparison of sonicated and boiled crude expression cell lysates and purified amidase by SDS-PAGE (left) and by zymogram (right) analysis. The letters are indicating that the same samples were added to both gels in the corresponding lanes. Expressing BL21 cells were harboring the orf27-pRSET A plasmid. (a) Sonicated crude BL21 cells, boiled, with 5x sample buffer. (b) Column purified endolysin, boiled, with 5x sample buffer. (c) Sonicated crude BL21 cells, not boiled, with 5x sample buffer. (d) Column purified endolysin, not boiled, with 5x sample buffer. (e) Column purified endolysin, not boiled, without 5x sample buffer. SDS-PAGE revealed the presence of a protein, slightly below 60 kDa (black arrow is showing 60 kDa on the protein ladder), regardless of the sample preparation method. In spite of the R4lys protein’s presence, zymography only showed its cell wall destroying amidase activity when the sample was not boiled (white bands in lanes (c, d, e)).