Fig. 5

In vitro characterisation of a NetB-C. jejuni heptasaccharide glycan conjugate expressed from an inducible promoter in E. coli CLM24 cedA::pglB strain and χ7122 pgl. a Crystal structure of pore-forming toxin NetB in its heptameric form (PDB: 4H56) [61]; b Schematic linear structures of NetB monomers modified to be suitable PglB substrates. G-NetB(10) is a glycosylatable toxoid version N-terminally fused to a PelB leader peptide for translocation to the periplasm, containing ten glycosylation sequons, and a C-terminal 6xHis tag. unG-NetB is the non-glycosylatable counterpart where each sequon has a NQ mutation abrogating N-linked glycosylation; c Western blotting showing results of a His-pulldown of G-NetB(10) and unG-NetB from E. coli glycoengineering strain CLM24 cedA::pglB containing a chromosomal copy of PglB and a plasmid encoding the pgl genes necessary to produce C. jejuni heptasaccharide (pACYCpglΔpglB) in lanes 1–2, and χ7122 pgl integrant in lanes 3–4. Percentage in parenthesis indicates sample loading (v/v) per lane; d dot-blot of OD_600nm normalised cells washed and resuspended in PBS shows G-NetB(10) glycoconjugate is exposed on the cell surface in CLM24 cedA::pglB, O-antigen negative and WaaL negative glycoengineering strain carrying pACYCpglΔpglB. IPTG induction that promotes the expression of both PglB and G-NetB(10) in this strain is indicated with + , if present, or −, if absent