Expression and purification of anthrax toxin protective antigen from Escherichia coli.
Anthrax toxin consists of three separate proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA binds to the receptor on mammalian cells and facilitates translocation of EF or LF into the cytosol. PA is the primary component of several anthrax vaccines. In this study we expressed and purified PA from Escherichia coli. The purification of PA from E. coli was possible after transporting the protein into the periplasmic space using the outer membrane protein A signal sequence. The purification involved sequential chromatography through hydroxyapatite, DEAE Sepharose CL-4B, followed by Sephadex G-100. The typical yield of purified PA from this procedure was 500 microg/liter. PA expressed and purified from E. coli was similar to the PA purified from Bacillus anthracis in its ability to lyse a macrophage cell line (J774A.1). The present results suggest that a signal sequence is required for the efficient translocation of PA into E. coli periplasmic space.
Sharma
M
M
Genetic Engineering Division, Centre for Biochemical Technology, Delhi, India.
Swain
P K
PK
Chopra
A P
AP
Chaudhary
V K
VK
Singh
Y
Y
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial; D001408:Bacillus anthracis; D001427:Bacterial Toxins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification; D017384:Sequence Deletion / Q000235:genetics
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D009943:Organophosphorus Compounds; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C030325:anthrax toxin; C008705:octamethyl pyrophosphoramide