Expression and purification of the Bacillus anthracis protective antigen domain 4.
The protective antigen (PA) of Bacillus anthracis plays a crucial role in the pathogenesis of the anthrax disease. The fourth domain of PA (PA-D4) is responsible for initial binding of the anthrax toxin to the cellular receptor, and thus, is an attractive target for structure-based drug therapies. A synthetic gene for PA-D4 has been prepared by recursive PCR. PA-D4 has been expressed as a fusion protein in Escherichia coli. PA-D4 has been purified to near homogeneity and its identity has been verified by mass spectrometry. The recombinant PA-D4 exhibits CD and NMR spectra that suggest that it is folded and amenable for biophysical studies. Moreover, recombinant PA-D4 binds to HeLa cells, which suggests that recombinant PA-D4 is functional to bind to its cellular receptor.
Krishnanchettiar
Sangeeth
S
Department of Biochemistry and Molecular Biology, University of Illinois at Chicago, Chicago, IL 60612, USA.
Sen
Jayita
J
Caffrey
Michael
M
D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000737:chemistry* / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry* / Q000302:isolation & purification; D001483:Base Sequence; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C030325:anthrax toxin