FIG 6.

Detailed analysis of aglycosylated LST variants. (A) SDS-PAGE analysis of enzyme expression and purification. Lanes: M, marker; 1, 10 μl of supernatant from shake flask culture; 2, 10 μl of supernatant from bioreactor culture; 3, 10 μg of purified protein. (B) Kinetic analysis of S. aureus lysis in microplate turbidometric assays. The horizontal line provides a visual guide to assess TOD₅₀. (C) Antibacterial efficacy as measured by MIC assay. An S. aureus laboratory strain (SA113) and three clinical isolates were assessed for growth inhibition by serial dilutions of purified enzymes. The OD₆₅₀ readings after 24 h of growth show that, while the different strains exhibited different MIC₀ values (P = 0.0008, two-way analysis of variance), wild-type LST and the engineered variants did not exhibit significantly different MIC₀s for any given strain (P = 0.8281), and neither was there a significant interaction between the different enzyme treatments and different strains (P = 0.6529). (D) Melting temperature analysis by differential scanning fluorimetry. The engineered aglycoslyated variants exhibit a single transition, whereas commercial wild-type LST exhibits two distinct transitions, one at a substantially lower temperature. Error bars represent standard deviations from three experiments.