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‘United States Patent: 6,387,665 @ Page 9 of 11 ‘same conditions and purity of the fractions was reassessed as described above. All fractions of greater than 95% purity were ultimately combined, aliquoted and frozen at -70.degree. C. subsequent to determination of the total PA concentration. Analysis and characterization of purified 83 kDa PA: Purified PA was quantitated by measuring UV- absorption at 280 nm using the relationship of 1 A.sub.280nm in a 1 cm pathlength cuvette is equals 1 mg PA/ml (Leppla, 1988). Results obtained in this manner were confirmed using the Bio-Rad Bradford protein assay under conditions suggested by the manufacturer. PA purity was assessed using SDS- PAGE under conditions described above. Capillary electrophoresis analytical assays have also proven promising in the assessment of PA purity and amounts of residual protease inhibitors in final product. Feasibility studies using a 47 cm.times.50 pm uncoated silica capillary and borate/SDS/acetonitrile buffer revealed an excellent separation of the protein from residual protease inhibitors. Quantitation of both protein and inhibitors has also proven possible, but the technique remains limited by the relatively high limits of detection (1 mM EDTA, 0.1 mM PMSF, and 0.05 mM OP) under current conditions. Automated N-terminal sequencing was carried out with purified PA using an Applied Biosystems 470A sequenator after desalting over Bio-Rad PD10 columns equilibrated with 5 mM NaCl and 1 mM CaCl.sub.2. A unique N-terminal sequence was found and the first six residues of the sequence were identical to PA from the endogenous plasmid pXO1 harbored by the USAMROD B. anthracis Sterne strain. In addition, the sequence corresponded exactly with the published DNA derived protein sequence (Welkos et al.). Native gel electrophoresis under non-denaturing conditions revealed that PA purified from .DELTA.Sterne-1(pPA102)CR4 also exhibited the microheterogeneity noted previously for PA produced by the Sterne strain. Cytotoxicity assays of the product using the macrophage lysis assay (Friedlander et al.) revealed that the titration curve of biological activity for PA from .DELTA.Sterne-1 (pPA102)CR4 was indistinguishable from that generated for PA from the Sterne strain. Evaluation of .DELTA.Sterne-1(pPA102)CR4: EXAMPLE 1 B. Anthracis .DELTA.Sterne-1(pPA102)CR4 was compared with its parent spore-forming strain B. anthracis .DELTA.Sterne-1(pPA102). Both organisms were plated onto sheep blood agar (a preferred medium for promoting bacterial spore production) and grown at 37.degree. C. for 1 day, after which the temperature was lowered to 25.degree. C. for 4 days. The two strains were also grown in liquid Leighton-Doi medium, which is designed to promote spore production, for 1 day at 37.degree. C. followed by 4 days growth at 25.degree. C. Growth from both agar and broth cultures were examined under phase contrast microscopy for the presence of spores. Growth from all four cultures were then resuspended in phosphate buffered saline to a concentration of about 10.sup.9 colony-forming units (CFU) per ml. All four cultures were then heat shocked at 64.degree. C. for 60 minutes to kill vegetative cells. Aliquots of 0.1 ml of the heat shocked material was then plated out onto sheep blood agar and incubated at 37.degree. C. for 2 days. Results: B. anthracis .DELTA.Sterne-1(pPA102): Spores were seen under microscopic examination of material from both the sheep blood agar cultures and the Leighton-Doi medium cultures. On sheep blood agar plates containing heat shocked culture material from both sheep blood agar cultures and Leighton-Doi medium cultures, there was confluent growth. The data clearly indicate that B. anthracis .DELTA.Sterne-1(pPA102) forms spores. B. anthracis .DELTA.Sterne-1(pPA102) CR4: No spores were seen under microscopic examination of material from both the sheep blood agar cultures and the Leighton-Doi medium cultures. On sheep http://patft.uspto.gov/netacgi/nph-Parser?Sectl=PTO2&Sect2=HITOFF &u=/netahtml/sear... 6/28/2005