Fig. 4

Formation of intramolecular disulfide bond of BLP. The B. subtilis ΔbdbDC strain and the parental strain 168 were transformed with pHY300PLK (empty vector) or pHY-BLP02. Cells were grown at 30 °C for 72 h in modified 2 × L-Mal medium. A SDS-PAGE analysis of culture supernatants. The supernatants were diluted two-fold with SDS reducing buffer. Five microliters of each sample were applied to SDS-PAGE. The expression plasmids and hosts are indicated above and below the image, respectively. The arrow indicates the position of mature BLP. B Pentaglycine cleavage activity assay of culture supernatants using the pentaglycine-containing FRET substrate FRET-GGGGG. Since the activity detected in the ΔbdbDC strain was very low, it was also included in the inset. The results presented are the means of three individual experiments. Error bars represent the standard errors of the means. C Non-reducing and reducing SDS-PAGE analysis of the purified BLP. The purified BLP was applied to SDS-PAGE at 500 ng/lane under non-reducing (lane 1) or reducing (lane 2) conditions