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United States Patent: 6,316,006 ® @ Page 8 of 10 * a linear gradient to 30% of the 1 M NaCl DEA elution buffer. The purified PA was found to elute between 10-15%, while the smaller molecular weight proteolytic breakdown products eluted as a shoulder or partially resolved peak at 16-20% of the elution buffer. The resolution of the two peaks was found to be a function of content of PA proteolytic degradation products. The eluant was monitored at 280 nm and peak fractions were collected by manual triggering of an ISCO fraction collector. Samples of the peak fractions were diluted into 5-10 volumes of TRIS pH8.0, 0.1 mM PMSF, 50 .mu.M OP, 1 mM EDTA buffer and concentrated using Amicon Centricon 30 concentrators at 4500.times.g at 4.degree. C. to approximately the initial sample volume. An equal volume of SDS-PAGE solubilization buffer was added to the sample immediately prior to heating at 95.degree. C. for 5 min. Purity was assessed from 8-25% SDS-PAGE PHAST gels (Pharmacia) and fractions with the highest purity were combined and dialyzed against 40-50 volumes of 25 mM DEA pH8.9, 50 mM NaCl, 0.1 mM PMSF and 2mM EDTA at 4.degree. C. for at least 16 hrs. Fractions judged empirically to be less than 95% pure were rechromatographed under the 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 .mu.m 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 NaC] 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 pX01 harbored by the USAMRID 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 mal of the heat shocked material was then plated out onto sheep blood agar and http://patft.uspto.gov/netacgi/nph-Parser?Sectl=PTO2&Sect2=HITOFF&u=/netahtml/sear... 6/28/2005