Identification of O-glycosylation sites in Clostridium botulinum and Geobacillus kaustophilus flagellins

In this study, we identified the O-glycosylation sites in recombinant Clostridium botulinum flagellin (CbFla) and Geobacillus kaustophilus flagellins, GkFlaA1 and GkFlaA2, expressed from the E. coli strains, EV136 and EV240 (K1:K12 strains engineered by Prof. Eric Vimr’s group to accumulate CMP-sialic acid in the cytosol), EV36 (wildtype K1:K12 strain), and BL21(DE3) (laboratory strain used for protein expression using T7 RNA polymerase). The recombinant flagellins were expressed with or without co-expression of motility associated factor (Maf) (flagellin nonulosonic acid glycosyltransferase) in these strains, purified and subjected to MS/MS analysis at Taplin Biological Mass Spectrometry facility.