Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis.
We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.
Rhie
Gi-Eun
GE
Department of Microbiology, National Institute of Health, 194 Tongil-Lo, Seoul 122-701, Korea.
Park
Young-Mia
YM
Chun
Jeong-Hoon
JH
Yoo
Cheon-Kwon
CK
Seong
Won-Keun
WK
Oh
Hee-Bok
HB
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000633:toxicity; D016863:Bacillaceae Infections / Q000276:immunology / Q000517:prevention & control; D001407:Bacillus / Q000235:genetics* / Q000276:immunology*; D001408:Bacillus anthracis / Q000235:genetics* / Q000276:immunology*; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000633:toxicity; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005260:Female; D015870:Gene Expression; D005798:Genes, Bacterial; D006168:Guinea Pigs; D066298:In Vitro Techniques; D051379:Mice; D009500:Neutralization Tests; D010957:Plasmids / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity; D013171:Spores, Bacterial / Q000235:genetics / Q000276:immunology; D014774:Virulence
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C030325:anthrax toxin