Rapid purification of recombinant anthrax-protective antigen under nondenaturing conditions.
Anthrax-protective antigen is the central moiety of the anthrax toxin complex that mediates the entry of the other two toxin components, lethal factor and edema factor into the cells. It is also the main immunogen of the cell-free vaccine against anthrax. However, in addition to PA, the vaccine contains trace amounts of other culture-derived proteins that contribute to the side effects of the vaccine like pain, edema, erythrema, etc. Thus there is a need to develop high-resolution purification methods to purify PA to homogeneity. In this study we have presented a purification strategy for rapid purification of recombinant protective antigen under nondenaturing conditions, which ensures that not only biological activity but also the conformational integrity of immunological epitopes is well-preserved. The protein was purified to homogeneity in a two-step purification procedure that takes just 6 h for completion. Three milligrams of recombinant protective antigen obtained from 1-liter culture was comparable to B. anthracis protective antigen in terms of functional and biological activity. Moreover, the immunogenicity elicited by the purified protein in mice was also studied. The studies reported here are part of continuing research that aims to provide a safe and efficacious alternative to the current vaccine against anthrax.
Ahuja
N
N
Centre for Biotechnology, Jawaharlal Nehru University, New Delhi-110067, India.
Kumar
P
P
Bhatnagar
R
R
D000818:Animals; D000942:Antigens, Bacterial / Q000302:isolation & purification* / Q000378:metabolism; D001408:Bacillus anthracis / Q000276:immunology*; D016466:CHO Cells; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D000942:Antigens, Bacterial; D011994:Recombinant Proteins; D000242:Cyclic AMP