Fig. 1

SDS-PAGE (a) and immunoblot analysis (b) of WbgUTEV6H expression in E. coli and purification of WbgU. E. coli SHuffle^® T7 Express cells carrying plasmids pET23a(+)_wbgUTEV6H and pMJS9 were grown in TB medium at 30 °C until OD₆₀₀ of 1 was reached. l-arabinose was added to a final concentration of 0.5% and the culture was incubated for another 6.5 h. The cells were harvested by centrifugation, lysed, and insoluble particulate fractions (IS) and soluble fractions (S) were isolated. WbgUTEV6H was purified from the soluble fraction using Ni–NTA chromatography (Ni–NTA 1). Fractions of the flow-through (FT), washing (W), and elution (E1–E6) were collected. Fractions E2–E6 were pooled (P) and incubated with TEV protease (P + T). The cleaved His-tag, TEV protease, and undigested WbgUTEV6H were removed using a second affinity chromatography step (Ni–NTA 2, E). Aliquots (10 μl) of the samples and collected fractions were separated by SDS-PAGE and visualized by Coomassie staining (a) and immunoblotting (b). WbgUTEV6H with an estimated mass of 41 kDa was detected using mouse Anti-His.H8 antibodies. Tag-free WbgU (40 kDa) was clearly visible in the flow-through (a, F1 and F2). The respective bands were no longer detected in the immunoblot assay with Anti-His.H8 antibodies (b, F1 and F2) indicating the successful removal of the polyhistidine tag. Molecular mass markers (MW) are in kDa