Fig. 2From: The YoaW signal peptide directs efficient secretion of different heterologous proteins fused to a StrepII-SUMO tag in Bacillus subtilisIdentification of signal peptide sequences for enhanced secretory expression of SUMO-fused heterologous proteins in Bacillus subtilis. a Visualisation of alkaline phosphatase activity of representative B. subtilis KO7A colonies on solid medium containing the chromogenic substrate BCIP and IPTG as an inductor of PhoA reporter-gene expression. The colonies derived from a transformation of B. subtilis KO7A with plasmid pJHS03 with the SPamyQ encoding sequence replaced by a DNA-library of 173 different B. subtilis signal peptide encoding sequences. b Extracellular PhoA activities of three B. subtilis KO7A clones isolated from the signal peptide screen. KO7A/pJHS02 served as a negative control (−), KO7A/pJHS03 (SPamyQ) and KO7A/pJHS04 (SPaprE) served as positive controls. The signal peptides were identified as SPyopL (pJHS05), SPyncM (pJHS06) and SPyoaW (pJHS07) by DNA sequencing of re-isolated plasmids. IPTG was added to each 50 ml bacterial culture shaking at 37 °C at a final concentration of 1 mM at an OD600 of 0.8. Samples were withdrawn after 4 h and PhoA activities were measured as described in “Methods” section. c In parallel to PhoA activity estimation (see above), the cell free culture supernatants were subjected to protein precipitation with trichloroacetic acid. Precipitated proteins were dissolved in one tenth of the volume of Laemmli-buffer and 10 µl were analysed via SDS-PAGE. The StrepII-SUMO-PhoA protein is labelled with a star (*). d Western blot analysis of culture supernatants (CS) precipitated with TCA and whole-cell lysate samples (CE). Blots were developed with anti-PhoA antibodies. CS: 10 µl of a 10-fold dilution of the samples used for Coomassie staining (see above) were loaded; CE: 10 µg whole-cell protein per laneBack to article page