FIGURE 2.

Experimental validation of Atu8019 lipidation. (A) Atu8019 protein sequence and predicted motifs/domains. (B) Schematic illustration of the lipoprotein labeling approach. (C) E. coli BL21 expression cultures containing the pET24 empty vector (EV) or producing one of the Atu8019^HIS derivatives (WT or C22A) were grown over night with alkyne-palmitic acid. After labeling, cells were harvested, lysed, and a copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC)-reaction with Azide-Fluor488 (AF488) was performed for in-gel fluorescence profiling of lipoproteins (top). The arrows indicate acylated Atu8019 protein before and after signal-peptidase II cleavage. Atu8019^HIS production was verified by Western blot (middle) and the coomassie blue-stained gel served as loading control (bottom). PS, protein standard.