Fig. 4From: The YoaW signal peptide directs efficient secretion of different heterologous proteins fused to a StrepII-SUMO tag in Bacillus subtilisGFP-specific scFv purification and antibody-antigen binding assay. a SDS-Page representing purification steps for the GFP-specific scFv antibody fragment from culture supernatants of B. subtilis strain KO7A/pJHS11, including immobilized metal ion affinity (IMAC) purification via the polyhistidine-tag and NiNTA, affinity purification via the StrepII-tag and StrepTactin agarose, and removal of the StrepII-SUMO part via SenP protease treatment. Cells were induced with IPTG at an OD600 of 0.8 and incubated for further 14 h at 30 °C. StrepII-SUMO-scFv fusion protein is marked with one star (*), GFP-specific scFv is marked with two stars (**), and StrepII-SUMO is marked with three stars (***). Lane 1 and 2: culture supernatant concentrated by TCA precipitation before (B) and after (A) NiNTA-agarose treatment. Lane 3: sample of wash fraction from first IMAC (W); lane 4: sample of elution fraction from first IMAC (E); lane 5: sample of elution fraction from StrepII-tag affinity purification; lane 6: sample of elution fraction from StrepII-tag affinity purification incubated with SenP-protease for 1 h at 37 °C; lane 7: sample of flow-through fraction of second IMAC (F); lane 8: sample of elution fraction from second IMAC (E). For details, see “Methods” section. b SDS-Page analysis of pull down assay using NiNTA magnetic agarose beads. GFP-specific scFv antibody fragments were bound to magnetic beads via the C-terminal polyhistidine tag (lanes are marked with a plus: +). Unloaded magnetic beads served as a control (marked with a minus: −). GFP was then added to the magnetic beads in excess as a purified native protein (lanes 3–6) or as a cleared lysate from E. coli cells overproducing GFP (lanes 7–10). As a negative control, no GFP was added (lanes 1 and 2). After 1 h incubation at 4 °C allowing interaction complex formation, the magnetic beads were removed, and a sample of each remaining supernatant (S) submitted to SDS-Page (lanes 1, 3, 5, 7 and 9). The beads were then washed and protein was eluted using 250 mM imidazole. The elution fractions (E) were submitted to SDS-Page (lanes 2, 4, 6, 8 and 10). GFP-specific scFv is marked with two stars (**)Back to article page