Fig. 3

Increasing Und-P levels promotes recombinantly expressed S. pneumoniae capsular polysaccharide in E. coli. (A and C) Western blots showing that increasing Und-P levels enhances production of S. pneumoniae capsule serotype 4 in E. coli cells harboring plasmid pB4 (contains capsule loci). Cells with the indicated genotypes were grown at 28 °C in 2YP media for 5 or 25 h. 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. Results are representative of three independent experiments. (B and D) ELISA. Capsular polysaccharide quantitated from cells grown in panels A and C. All experiments represent three biological replicates, with each sample probed in duplicate. Values are expressed as means of ng capsular polysaccharide serotype 4 production per µl of culture adjusted to OD₆₀₀ = 10. Error bars show standard errors of the means. Significance was determined using an ordinary one-way ANOVA with Dunnett’s multiple correction. Asterisks above sample bars denote significance relative to CLM37/pB4/pgne/vector. *p < 0.05, **<0.005, ***<0.001, **** <0.0001. Absolute capsular polysaccharide values are detailed in Table 1. The E. coli strains shown are EJK1 (W3110/pB/puppS), EJK2 (W3110/pB4/vector), EJK3 (W3110/pB4/puppS), EJK7 (CLM37/pB4/pgne/vector), EJK8 (CLM37/pB4/pgne/puppS), EJK11 (ΔPGT/GT/pB4/pgne/vector), and EJK12 (ΔPGT/GT/pB4/pgne/puppS).