Purification of cell wall fragments (CWF) and coating of CWF by recombinant S-layer proteins
Cell wall fragments were purified from L. amylovorus cells as described earlier [33]. Purified cell walls were lyophilized and stored as suspensions in water at -20°C. In order to coat the cell walls, the affinity purified recombinant S-layer proteins were dissolved in 5 M GuHCl at a concentration of 30 μg/ml, dialyzed against 50 mM Tris–HCl (pH 7.0) at +4°C overnight and centrifuged (40,000 g, 30 min, +4°C) to remove large protein aggregates. The protein concentrations of the supernatants were determined by the Bradford method, immediately after which the supernatant proteins and the cell walls were combined in a ratio 1:4 (W/W) and incubated overnight at +4°C with rotation. The coated CWF were collected by centrifugation (25,000 g, 30 min, +4°C), resuspended into DMEM/Ham’s F-12 [1:1] medium and analyzed by SDS-PAGE. To verify the absence of large protein aggregates among the coated CWF, the preparations in 50 mM Tris–HCl (pH 7.0) were routinely negative stained by uranyl acetate (5 min on ice) and observed by JEOL 1200-EX II transmission electron microscope at the operating voltage of 80 kV.