Fig. 1. Proteomic identification of a spore coat-associated protein and its similarity with camelysin. Protein (300 μg) from dormant spores of the Bacillus anthracis Sterne strain was separated in a silver-stained 2-DE gel as described in Materials and methods (a). A circled spot was subsequently excised and analyzed by in-gel trypsin digestion followed by MALDI-TOF MS. The MALDI-TOF MS spectrum (b) of a circled spot revealed that the tryptic peptide mass fingerprint with eight peptide masses match with that of spore coat-associated protein (SCAP) in Bacillus anthracis (Accession No. Q81TI3). The predicted peptide fragments corresponding to observed m/z values observed for SCAP are shown (b). The tryptic autodigestive peak at m/z value 2164.04, indicated by an asterisk, served as an internal calibration standard. The amino acid homology of SCAP (blue) to camelysin (black) (Accession No. Q63E88) in Bacillus cereus was illustrated (c). Asterisks indicated the amino acid identities. Amino acid residues (9–29) were predicted as a transmembrane domain (red).

Fig. 2. Confirmation of expression and purification of SCAP. The construct encoding SCAP for protein expression was described in Materials and methods. SCAP was expressed in the E. coli in the absence (a, lane 1) or presence (a, lane 2) of 1 mM IPTG. After IPTG induction, SCAP was successfully expressed in E. coli and shown by 12.5% SDS–PAGE (arrow). Purified SCAP at about 21 kDa (a, lane 3, arrowhead) was obtained by using Ni-NTA agarose. Purified SCAP was obtained by removing the SCAP-tag with factor Xa protease. The expression and purity of SCAP were confirmed by analysis of spectra of MALDI-TOF MS (data not shown) and Q-TOF MS/MS sequencing (b). The MALDI-TOF MS spectrum of purified SCAP is similar with that in Fig. 1b. An internal peptide of SCAP with m/z value at 1008.46 analyzed by MALDI-TOF MS was sequenced by Q-TOF MS/MS as FLWNWDK (b).

Fig. 3. SCAP triggers cells in bronchoalveolar larvage fluid to undergo apoptosis. Purified SCAP (6.26 ng) was injected into the lung of A/J mice via intra-tracheal instillation. One day after injection, cells from bronchoalveolar lavage fluid were isolated and examined by TUNEL assay. Cells isolated from A/J mice treated with PBS (A) or proteins purified from E. coli transformed with an empty expression vector (B, vector) showed no apoptotic cells. In contrast, apoptotic cells (arrows) are detectable in bronchoalveolar lavage fluid isolated from mice injected with purified SCAP for one day (C). Light blue indicates the merging of green fluorescein-12-dUTP with nuclei dye Hoechst 33258, representing the localization of apoptotic cells. Bar, 5 μm.

Fig. 4. UV-irradiation of an E. coli vector-based vaccine over-expressed SCAP. Bacteria were plated on a LB-agar plate as indicated (a). The upper half of the plate is control bacteria that were transformed with empty expression vectors. The lower half is the colonies of SCAP over-expressed E. coli. The left and right halves of the plate are bacteria without (−UV) and with UV irradiation (+UV), respectively. The result indicates either control or SCAP-over-expressed bacteria are efficiently inactivated by 3500 J/m² of UV irradiation. Protein profile in bacteria transformed by a SCAP fusion protein expression plasmid before (lane 1; −IPTG) and after (lane 2 and 3: +IPTG) IPTG induction is shown on a Coomassie blue gel (b). There was no significant change of protein expression profile including SCAP (arrow) before (lane 2) and after (lane 3) UV-irradiation.

Fig. 5. Anti-SCAP antibody assays. Antigens indicated were spotted twice for each dilution of 10-fold serial dilution (a). The upper and lower arrays were hybridized with sera (1:250 dilution) from mice immunized with an empty expression vector (Control) or a SCAP over-expressing E. coli vector (SCAP). Arrays were spotted with MBP-tagged SCAP (MBP-SACP; enclosed by white rectangles), SCAP (enclosed by pink rectangles) and mouse IgG (enclosed by yellow rectangles). Mouse IgG is used as a positive reaction as well as a reference for array normalization. The digital images in panel A were quantified and analyzed with GenePix 6.0, Cluster and TreeViw programs (b). The spot intensities were normalized to the average of two spots of IgG with 100× dilution on each array. The product value of each spot was expressed in a gray-scale fashion. The result unambiguously revealed anti-SCAP antibody production from the SCAP immunized mice. Factor Xa protease cleaved SCAP was subjected to SDS–PAGE, transferred to a nitrocellulose membrane and blotted with immunized mouse sera (c). A band at 21 kDa, consistent with full-length SCAP protein was recognized by sera (1:500 dilution) from SCAP (right) immunized but not control (left) mice. Two identical samples were run on the gel for each blot.