High level constitutive production of anthrax protective antigen

• Main Authors: BHATNAGAR RAKESH, CHAUHAN VIBHA, MOHSIN WAHEED SAYED
• Format: Patent
• Language: English
• Published: 12.02.2008
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS THEREOF; CULTURE MEDIA; ENZYMOLOGY; FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIREDCHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERSFROM A RACEMIC MIXTURE; HUMAN NECESSITIES; HYGIENE; MEDICAL OR VETERINARY SCIENCE; METALLURGY; MICROBIOLOGY; MICROORGANISMS OR ENZYMES; MUTATION OR GENETIC ENGINEERING; ORGANIC CHEMISTRY; PEPTIDES; PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES; PROCESSES USING MICROORGANISMS; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS ORMEDICINAL PREPARATIONS; SPIRITS; VINEGAR; WINE

The present invention relates to a process for preparing anthrax protective antigen protein from E. coli using fed batch culture. This process creates a constitutively expressing system for rapid, efficient, cost-effective and high-level production of anthrax PA from E. coli. The steps of the process involves, transforming E. coli DH5alpha cells with the recombinant constitutive expression plasmid containing the PA gene to obtain recombinant DH5alpha cells and testing the PA expression by lysis of said cells followed by denaturing gel electrophoresis and Western Blotting technique using PA antibodies. This is followed by fermentation and harvesting of the high cell density cells. The said cells are solubilized using 6-8 Molar Urea and separated by centrifugation. The urea denatured PA is isolated from said supernatant and purified and thereafter eluted.