Fig. 4

Increasing Und-P levels increases glycan expression in E. coli cells engineered for enhanced expression of S. pneumoniae capsular polysaccharides. Western blots showing that increasing Und-P levels by overexpressing uppS enhances expression of S. pneumoniae capsule serotype 4 (expressed from pB4) in E. coli Sparrowhawk cells over shorter periods of time. Cells were grown at 28 °C in 2YP media containing 500 µM IPTG (0 µM IPTG for baseline [BL]) for 7.5 h (panel A) or 23 h (panel B). Lysed, whole cell samples were then separated by SDS-PAGE on a 4–12% bis-tris gel and detected using anti-serotype CPS primary and anti-rabbit fluorescent secondary antibody. The E. coli strains shown are EJK13 (pB4), EJK14 (pB4/vector), and EJK15 (pB4/puppS).