Expression and purification of the recombinant protective antigen of Bacillus anthracis.
Protective antigen (PA) is a major component of the vaccine against anthrax. The structural gene for the 83-kDa PA was expressed as fusion protein with 6x Histidine residues in Escherichia coli. Expression of PA in E. coli under the transcriptional regulation of the T5 promoter yielded an insoluble protein aggregating to form inclusion bodies. The inclusion bodies were solubilized in 6 M guanidine-HCl and the protein was purified under denaturing conditions using nickel nitrilotriacetic acid (Ni-NTA) affinity chromatography. The denatured protein was renatured by gradual removal of the denaturant while immobilized on the Ni-NTA column. The protein was then purified using Mono-Q column on FPLC. The yield of the purified recombinant PA (rPA) from this procedure was 2 mg/liter of the culture. The rPA had an apparent molecular mass of 83 kDa as determined by SDS-PAGE. Antisera to native PA recognized the fusion protein. The rPA was biologically as well as functionally active. Thus, the recombinant PA may be used to develop an effective recombinant vaccine against anthrax.
Gupta
P
P
Centre for Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India.
Waheed
S M
SM
Bhatnagar
R
R
D000818:Animals; D000915:Antibody Affinity; D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000737:chemistry* / Q000276:immunology*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification*; D002460:Cell Line; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004306:Dose-Response Relationship, Immunologic; D004926:Escherichia coli / Q000235:genetics; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014614:Vaccines, Synthetic / Q000737:chemistry / Q000276:immunology
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; C030325:anthrax toxin