Intoxication of mammalian cells by Bacillus anthracis requires the coordinate activity of three distinct bacterial proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). Among these proteins, PA has become the major focus of work on monoclonal antibodies and vaccines designed to treat or prevent anthrax infection since neither EF nor LF is capable of inducing cellular toxicity in its absence. Here, we present the development of a sensitive, precise, and biologically relevant bioassay platform capable of quantifying antibody-mediated PA neutralization. This bioassay is based on the ability of PA to bind and shuttle EF, a bacterial adenylate cyclase, into mammalian cells leading to an increase in cAMP that can be quantified using a sensitive chemiluminescent ELISA. The results of this study indicate that the cAMP-induction assay possesses the necessary performance characteristics for use as both a potency-indicating release assay in a quality control setting and as a surrogate pharmacodynamic marker for ensuring the continued bioactivity of therapeutic antibodies against PA during clinical trials.

Anthrax is caused by the gram-positive, spore-forming bacterium, Bacillus anthracis. The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor. Current vaccines against anthrax use PA as their primary component since it confers protective immunity. In this work, we expressed soluble, recombinant PA in relatively high amounts in the periplasm of E. coli from shake flasks and bioreactors. The PA protein was purified using Q-Sepharose-HP and hydroxyapatite chromatography, and routinely found to be 96–98% pure. Yields of purified PA varied depending on the method of production; however, medium cell density fermentations resulted in approximately 370mg/L of highly pure biologically active PA protein. These results exhibit the ability to generate gram quantities of PA from E. coli.

The metallothionein-human growth hormone fusion gene (MThGH), constructed by fusing the mouse metallothionein promoter with the human growth hormone gene, was microinjected into the cytoplasm of one-cell embryos of channel catfish Ictalurus punctatus. The 3-week-old fish that developed were analyzed for integrated copies of the MThGH gene. Two of 10 animals contained MThGH sequences that comigrated with genomic DNA of high molecular weight in southern blots. The MThGH sequences were organized in head-to-tail tandem arrays that are characteristic of foreign genes integrated into recipient cell genomes. These data strongly suggest that the MThGH genes are stably integrated into chromosomal DNA, and that transgenic channel catfish can be generated by cytoplasmic injection of early embryos.