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Page 4 of 11 United States Patent: 6,387,665 * effects. The producing organisms of the invention are also, surprisingly, non-sporulating. Furthermore, the desired antigen is expressed into the supernatant. Hence, the protective antigen produced is easily purified and, though protective, does not cause many of the troublesome side effects of prior art vaccines. The organisms of the invention lacking spore-forming function may be killed by heat shock at temperatures as low as 60.degree. C. for 60 minutes. Hence, contamination of the environment with viable spore-forming organisms is easily avoided and decontamination is easily accomplished. Genesis of .DELTA.Sterne-1(pPA102)CR4: A 6kb Bam HI fragment harboring the PA structural gene isolated from the endogenous Sterne plasmid pXO1 was ligated into plasmid pBR322 and cloned into Escherichia coli bacteria (Vodkin and Leppla, 1983). From the resultant recombinant plasmid pSE36, the 6 kb fragment was then subcloned into the gram-positive vector pUB110 using the Bam HI restriction site. The resulting plasmid was transformed into B. subtilis IS53 and two stable PA producing, kanamycin resistant isolates were found (pPA101 and pPA102) (Ivins and Welkos, 1986). Subsequent analysis of the plasmids revealed that both had suffered spontaneous deletions. The pPA102 was found to have lost 4.2 kb of DNA from 363 bp 3' of the kanamycin resistance gene to approximately 164 bp 5' of the start of the PA structural gene, a result consistent with the observed inactivation of the phleomycin resistance gene of pUB110. The plasmid was then electrotransformed into .DELTA.Sterme-1, a plasmid-free strain of B. anthracis (Infection and Immunity, 52:454-458 (1986) and transformants were selected for kanamycin resistance. Transformants displaying a stable PA+, kanamycin resistant, (LF-, EF-, capsule-) phenotype were selected, This strain, .DELTA.Sterne-1(pPA102), was then subjected to Congo Red agar selection for mutants displaying an inability to bind the dye, a characteristic known to correlate with an asporogenic phenotype (Worsham, submitted). The selected isolate, now designated .DELTA.Sterne-1(pPA102)CR4 was further subcultured three times to insure that a single clone was isolated. This clone has served as the seed stock for all research and development of fermentation conditions, and purification of PA. Materials and Methods: Fermentation Conditions Media: FA medium was used for all plates and liquid cultures described here unless otherwise specified. FA medium consisted of 33 g/l tryptone (Difco), 20 g/l yeast extract (Difco), 2 g/l L-histidine, 8 g/l Na2HPO4, 7.4 g/l NaCl, 4 g/l KH2P04 adjusted to pH 7.4 with NaOH. Precultures: A working stock of .DDELTA.Sterne-1(pPA102)CR4 was prepared from the seed culture by streaking cells on an FA medium plate containing 40 .mu.g/ml of kanamycin. A sweep from the confluent growth zone on plate was cultured one time in liquid FA medium supplemented with kanamycin 40 .mu.g/ml to a final O.D..sub.600nm of 4.0. This culture was checked for purity by streaking on SBA plates, and diluted into multiple vials containing sterile 100% glycerol to a final glycerol concentration of 50% (v/v). These stocks were stored at -70.degree. C. A single vial was removed at the start of each fermentation cycle and discarded after use. The defrosted cells were streaked onto FA plates containing 40 .mu.g/ml kanamycin and incubated at least 16 hrs at 37.degree. C. After 16 hrs the plated cells were used to inoculate 50 mis of FA medium supplemented with 40 .mu.g/ml kanamycin in a 250 mi baffled-Erlenmeyer flask (Bellco Laboratories). The culture was incubated at 37.degree. C. at 200 rpm for 6 hrs or until an O.D..sub.600nm of 4-6 was obtained. The cells were then subcultured into 50 mls of FA medium in an identical flask under identical conditions. After 6 brs, or a culture O.D..sub.600nm of 6.2-6.5, a 1.6% (v/v) inoculum was transferred to 300 mls of PA medium supplemented with 40 .mu.g/ml kanamycin in a 2 liter baffled Erlenmeyer and incubated at 37.degree. C. at 200 rpm for 7 hrs, or until a final O.D..sub.600nm of 3.5-3.7 was achieved. http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF &u=/netahtml/sear... 6/28/2005