Fig. 3

In vitro activity of WbgU analysed by capillary electrophoresis (CE) (a) and in combination with GalNAc-T2 using a modified glycosyltransferase assay (b). Substrate conversion of the sugar substrates UDP-GlcNAc (blue squares) and UDP-GalNAc (yellow squares) using 0.15 mM (small squares) and 1.50 mM (big squares) in the presence of WbgU. UDP-GlcNAc and UDP-GalNAc were separated by CE and detected at 254 nm. The mean substrate conversion was calculated based on the relative peak area of three independent experiments and results are shown including the standard deviation (a). The mean specific activities of recombinant human GalNAc-T2 (rhGalNAc-T2) commercially produced in NS0 cells and His-tagged GalNAc-T2 (HisDapGalNAc-T2) expressed in E. coli were determined in four independent experiments as duplicates using 0.5 mM UDP-GalNAc and UDP-GlcNAc, as activated sugar donors, 0.25 mM EA2 peptide acceptor substrate and 1 µg WbgU. Samples containing WbgU were incubated for 20 min at 37 °C prior to glycosyltransferase addition and subsequently incubated for 20 min at 37 °C. Results are displayed including the standard error; inserted numbers indicate the specific activities in pmol*min⁻¹*µg⁻¹ (b). n.s. not significant