Fig. 4

Cleavage of surface-expressed SUMO-fused mCherry by surface-expressed Ulp1 and purification of mCherry. a SDS-PAGE analysis (left panel) and western blot (right panel) of surface-displaying bacterial cells and released target protein fractions. IPTG-induced E. coli cells (pET-LO-SUMO-mCherry/BL21 and pET-YfaL-Ulp1/BL21) were incubated together in cleavage buffer at 37 °C for 30 min, after which the cells and the buffer solution were immediately separated by centrifugation. Lane 4: cell pellets before incubation; lane 5: cell pellets after incubation; lane 6: supernatant fraction after incubation. The negative control (pET-LO-SUMO-mCherry/BL21 and pET/BL21) was prepared using the same procedures (Lane 1–3). Cleavage was conducted under different conditions of ionic strength (b), temperature (c), and incubation time (d) to optimize the cleavage efficiency and mCherry protein yield. The supernatant fraction after cleavage and centrifugation was analyzed by SDS-PAGE. e SDS-PAGE analysis of purified mCherry protein prior to (lane 1) or after (lane 2) ultrafiltration using a 30-kDa ultrafiltration tube. The results are representative of three independent experiments