Fig. 5
Analysis of purified AcrA-SP4 Glycoconjugates produced from different E. coli strains. A 5 µg protein separated by SDS-PAGE 4–12% bis–tris gel run with MOPS buffer. Glycan was detected using anti-serotype 4 primary antibody and anti-rabbit 800 fluorescent secondary antibody (green). Protein was detected using anti-His primary antibody and anti-mouse 680 fluorescent secondary antibody (red). M molecular weight marker PageRuler Plus. All strains contained plasmid pWA2 which expressed C. jejuni AcrA protein at 40 kDa. Presence or absence of pB4 recombinant S. pneumoniae serotype 4 capsule and cjpglB are denoted below the lanes as ± respectively, as are the strain names. In the case of cjpglB + P indicates the presence of plasmid pEXT22:pglB and + C indicates cjpglB integrated into the chromosome. For strain W311B cjpglB is under control of Ptac promoter, rather than the Anderson 0.1 promoter. B Quantification of capsular polysaccharide production by sandwich ELISA. Biological triplicate samples were processed in triplicate. Values for amount of capsule produced were interpolated from a standard curve of purified type-specific capsular polysaccharide (Statens Serum Institut, Denmark) and expressed as capsule produced per 2.5 µg of protein. Data are presented as the mean of biological replicates with error bars denoting the standard error of the mean. Significance was determined using a one-way ANOVA with Tukey’s multiple correction. Asterisks above sample bars denote significance relative to bar 8 (Sparrowhawk pWA2 pB4 pEXT22:PglB) **P < 0.005, ***P < 0.0005, ****P < 0.0001