Figure 6.

Solid-phase synthesis of CPSX oligosaccharides. A, schematic of the experimental setup. A reaction mix containing UDP-GlcNAc and primCPSX was circulated through a HisTrap column containing immobilized ΔN58ΔC98-CsxA, and an amylose column was linked to retain bled-off protein. B, UDP-GlcNAc consumption and parallel UMP production were determined by HPLC-AEC to follow the progress of the reaction. C and D, Alcian blue/silver-stained PAGE (C) and HPLC-AEC (D) were used to compare the dispersity of the enzymatically generated oligosaccharides to marker material from vaccine manufacturing. E, Western blot analysis developed against the N-terminal MBP tag (green channel) and the C-terminal His₆ tag (red channel) demonstrated that, at all sampled time points, the reaction mixture was free of protein, whereas elution of the HisTrap column with imidazole released the protein.