Figure 1.

SDS-PAGE and immunoblot analysis of HisDapGalNAcT2 expressed in E. coli . (A) Origami™ 2(DE3)pLysS cells carrying plasmid pET23d(+)::HisDapGalNAcT2 were grown in LB medium at 37°C until OD₆₀₀ 0.5, at which point IPTG (final concentration 1 mM) was added and cultures were incubated for a further 5 h. Cells were harvested by centrifugation, lysed and total cell lysate (T), the soluble protein fraction (S) and the insoluble particulate fraction (P) were separated by SDS-PAGE and visualised by Coomassie staining. HisDapGalNAcT2 with an estimated mass of 61.7 kDa (indicated by arrow) was not detected in the soluble (S) cell fraction, but a band of the correct size in the insoluble particulate (P) fraction (★) was excised and further analysed by ESI-MS. (B) SHuffle® T7 cells harbouring either pET23d(+)::HisDapGalNAcT2 in the absence or presence of pMJS9 were grown in EnPresso B medium. Fractionated cell samples were separated by SDS-PAGE and visualised by Coomassie staining (B) or immunoblotting (C) using a mouse anti human GALNT2 antibody. Molecular weight markers (MW) are in kDa. HisDapGalNAcT2 with an estimated mass of 61.7 kDa (arrow) was detected in soluble (S) and particulate (P) cell fractions. Commercially available rhGalNAcT2 (PC) and cell lysates of SHuffle® T7 pET23d(+) and SHuffle® T7 pMJS9 cells (NC) were included as controls.