Optimized production and purification of Bacillus anthracis lethal factor.
Bacillus anthracis lethal factor (LF) is a 90-kDa zinc metalloprotease that plays an important role in the virulence of the organism. LF has previously been purified from Escherichia coli and Bacillus anthracis. The yields and purities of these preparations were inadequate for crystal structure determination. In this study, the genes encoding wild-type LF and a mutated, inactive LF (LF-E687C) were placed in an E. coli-Bacillus shuttle vector so that LF was produced with the protective antigen (PA) signal peptide at its N-terminus. The resulting vectors, pSJ115 and pSJ121, express wild-type and mutated LF fusion proteins, respectively. Expression of the LF genes is under the control of the PA promoter and, during secretion, the PA signal peptide is cleaved to release the 90-kDa LF proteins. The wild-type and mutated LF proteins were purified from the culture medium using three chromatographic steps (Phenyl-Sepharose, Q-Sepharose, and hydroxyapatite). The purified proteins were greater than 95% pure and yields (20-30 mg/L) were higher than those obtained in other expression systems (1-5 mg/L). These proteins have been crystallized and are being used to solve the crystal structure of LF. Their potential use in anthrax vaccines is also discussed.
Park
S
S
Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, Bethesda, Maryland, 20892, USA.
Leppla
S H
SH
D019943:Amino Acid Substitution; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001407:Bacillus / Q000235:genetics; D001408:Bacillus anthracis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics; D014169:Transformation, Bacterial
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D021382:Protein Sorting Signals; C030325:anthrax toxin; D008666:Metalloendopeptidases