Proteolytic and Lytic Activities of VgrG2b

Generic protease activity of 10 μg recombinant VgrG2b_C-ter, its inactive mutant and a proteinase K control was assessed using 1% proteinaceous substrates bovine serum albumin and gelatin in 2% bacteriological agar plates. After incubation at 37°C for 24 h, plates were stained with amido black solution (Vermelho et al., 1996 (link)) to visualize zones of proteolysis. Lysozyme activity of recombinant proteins utilized the lyophilised substrate Micrococcus lysodeikticus (Sigma Aldrich) as previously documented (Lossi et al., 2011 (link)). The polymyxin B-mediated permeabilisation of the bacterial outer membrane to permit access of exogenous proteins to the periplasmic space was performed as described elsewhere (Brooks et al., 2013 (link)), except E. coli was employed. E. coli DH5α was resuspended in turbidometry buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl) at OD₆₀₀ 0.2 and incubated with 4 μg/ml polymyxin B. After addition of 10 μg purified protein, the turbidity at OD₆₀₀ was monitored at five-minute intervals for 1 h.