Fig. 6

Purification of T7Muc10 using Ni–NTA affinity chromatography. Cell pellets of E. coli SHuffle^® T7 Express carrying plasmid pMJS9 in combination with vector construct pET23d_galNT2_T7Muc10 or pET23d_galNT2_T7Muc10_wbgU expressing GalNAc-T2 and T7Muc10 or GalNAc-T2, T7Muc10 and WbgU were thawed and lysed. The soluble fractions were pre-purified using anion exchange chromatography (AIEX). Fractions of the flow-through containing T7Muc10 (AIEX) were loaded onto a Ni–NTA affinity chromatography spin column. Fractions of the flow-through (FT), the first washing step with equilibration buffer (W1), and the second washing step with washing buffer (W2) were collected. T7Muc10 was eluted by applying elution buffer twice (E1, E2). Aliquots of the fractions were analysed by SDS-PAGE stained with Coomassie (a) and Immunoblot treated with Anti-His.H8 antibodies (b). Molecular mass markers (MW) are in kDa