Expression and purification of functional human anthrax toxin receptor (ATR/TEM8) binding domain from Escherichia coli.
Anthrax is caused by the gram-positive, spore-forming bacterium, Bacillus anthracis. Anthrax receptors play a crucial role in the pathogenesis of the anthrax disease. Anthrax toxin receptor ATR/TEM8 VWA domain is responsible for the binding of protective antigen (PA) of B. anthracis, and thus an attractive target for structure-based drug therapies. However, the production of soluble and functional ATR/TEM8 VWA domain currently requires the use of mammalian expression systems. In this work, we expressed the ATR/TEM8 VWA domain as a fusion protein in Escherichia coli. Recombinant ATR/TEM8 VWA domain has been purified to homogeneity, and its identity has been verified by both N-terminal protein microsequencing and mass spectrometry. The purified ATR/TEM8 VWA domain exhibits very high affinity to PA based on BIAcore assay. Moreover, like the domain expressed in mammalian system, the bacterially expressed ATR/TEM8 VWA domain can block cytotoxicity induced by anthrax toxins, suggesting that the bacterially expressed ATR/TEM8 VWA domain is properly folded and fully functional.
Ding
Zhiping
Z
Leukocyte Biology and Inflammation Program, Renal Unit, Nephrology Division, Massachusetts General Hospital, Harvard Medical School, 149 13th street, Charlestown, MA 02129, USA.
Bradley
Kenneth A
KA
Amin Arnaout
M
M
Xiong
Jian-Ping
JP
D000818:Animals; D000942:Antigens, Bacterial / Q000378:metabolism; D001408:Bacillus anthracis / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000378:metabolism; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D008840:Microfilament Proteins; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020349:Surface Plasmon Resonance
C473287:ANTXR1 protein, human; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C030325:anthrax toxin