Fig. 3
SDS-PAGE analysis of scFv production in B. subtilis using the SUMO fusion system. a Comparison of secretory production of the StrepII-SUMO-scFv fusion protein with the AmyQ signal peptide (SPamyQ) and the YoaW signal peptide (SPyoaW). Liquid cultures in LB-medium were incubated in a 37 °C water bath under rigorous shaking. IPTG was added to a final concentration of 1 mM at an OD600 of 0.8, and cultures were incubated for further 4 h. Lanes 1–3: B. subtilis KO7A/pJHS08 (SPamyQ), lanes 4–6: KO7A/pJHS10 (SPyoaW), lanes 7–11: KO7A/pJHS11 (SPyoaW; SUMO and GFP-specific scFv encoding sequence codon optimized for B. subtilis). Lanes 1–9: cell free culture supernatant before (B) and after (A) incubation with NiNTA-agarose, and elution fraction (E) from NiNTA agarose. Lanes 10 and 11: NiNTA-agarose directly added to culture without removing cells by centrifugation. The StrepII-SUMO-scFv fusion protein is labelled with a star (*), and the amount of that protein purified via IMAC from 1 l of liquid culture is indicated below the lines of elution fractions (mg protein as estimated by a Bradford assay). b Analysis of secretory production of the StrepII-SUMO-scFv fusion protein with strain KO7A/pJHS11 (SPyoaW; SUMO and GFP-specific scFv encoding sequence codon optimized for B. subtilis) with cultivation at 30 °C and 37 °C, and IPTG induction for 4, 8, and 14 h. For further details see above