Patent ID: 7329513

Claim:
A process for preparing anthrax protective antigen protein from E. coli . using fed batch culture comprising the steps of (a) transforming E. coli DH5α cells with a recombinant constitutive expression plasmid containing the Protective Antigen gene to produce recombinant DH5α cells expressing the Protective Antigen protein, (b) growing said recombinant DH5α cells and testing Protective Antigen expression by lysis of said recombinant cells followed by denaturing gel electrophoresis and a Western Blotting technique using Protective Antigen antibodies, (c) fermenting said recombinant cells in a bio-reactor in Luria Broth medium at 32-42° C. in a fed batch culture, wherein the medium comprises nutrients, including a primary supplement selected from any one or all of a polyol, a carbohydrate and an organic acid, and wherein the fermenting comprises simultaneously monitoring a dissolved oxygen concentration and pH of the medium and, as nutrients are depleted from the medium, adding replacement nutrients to the medium to maintain the dissolved oxygen concentration and pH at levels that result in attainment of a high cell density culture that expresses Protective Antigen in a yield of at least 5 g/l, (d) harvesting said fermented cells by centrifugation of said high cell density culture at 5000-10,000 rpm for 10-30 minutes, (e) solubilizing said high cell density culture cells by using 6-8 Molar Urea solution and stirring at ambient temperature for 1-2 hours, (f) separating high cell density culture debris by centrifugation at 10,000-15,000 rpm for 30-60 minutes at 32-42° C. and collecting supernatant containing urea denatured Protective Antigen, (g) isolating said urea denatured Protective Antigen from said supernatant and purifying it by NI-NTA chromatography by gradual removal of urea with said Protective Antigen bound to an affinity column whereby to form purified renatured Protective Antigen, and (h) eluting the purified renatured Protective Antigen and, optionally, storing Protective Antigen protein as frozen aliquots at −20 to −70° C.