Fig. 5

Large-scale culture of transformed BL 21 (DE3) ΔA E. coli for preparing G-PEN-HuscFv34-6× His. (a) The percentage of intact bacterial cells (grown in 1000 mL broth for 5 h) that displayed the fusion protein on their surface as determined by flow cytometry (Q2 of the right panel). (b) Left sheet: Western blot patterns and intensities of G-PEN-HuscFv34-6× His bands (between 25 and 35 kDa, arrowhead) in supernatants of the bacteria in (a) after incubating in Sortase cleavage buffer for 0, 1, 2, 3, 4 and 5 h (lanes 1–6, respectively). Right sheet, Western blot patterns of the proteins in the supernatants that were bound by anti-SUMO (arrowhead); this band should be SUMO-Δ59 Sortase-LPET- that still linked to LPP′-OmpA′ on the membrane of lysed bacterial cells. (c) Left sheet: Lane 1 is SDS-PAGE and CBB stained pattern of the purified G-PEN-HuscFv34-6× His (arrowhead) from the supernatant of the transformed bacteria suspended in Sortase cleavage buffer for 2 h; middle and right sheet show Western blot patterns of the purified G-PEN-HuscFV34-6× His probed with anti-His antibody (lane 2) and mouse monoclonal anti-HuscFv34 (lane 3). Lanes M of (b) and (c) are protein molecular masses in kDa