Research Article

An O-Antigen Glycoconjugate Vaccine Produced Using Protein Glycan Coupling Technology Is Protective in an Inhalational Rat Model of Tularemia

Figure 1

Recombinant ExoA modified to incorporate additional glycosylation sequons is heavily glycosylated by F. tularensis O-antigen by C. jejuni PglB in E. coli CLM24. Samples were separated by SDS-PAGE, and then two-colour Western blots were used to simultaneously detect the degree of glycosylation of ExoA using (a) a monoclonal mouse antibody (FB-11) with specificity to F. tularensis O-antigen (red) and (b) rabbit polyclonal antibodies with specificity to the 6x His sequence (green). The two IR secondary antibody channels when overlaid (IR 800/680) result in images with overall yellow colour indicating conjugation (c). M, protein ladder marker; lane 1, pGVXN150 only; lane 2, pGVXN150 ExoA glycosylated with the F. tularensis O-antigen (same construct from Cuccui et al. [23]); and lane 3, GtExoA heavily glycosylated with the F. tularensis O-antigen due to the presence of an additional eight glycosylation sequons.