title: Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis.
abstract: Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGFα). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGFα). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.
authors.affiliation: National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Bachran
authors.forename: Suzanne
authors.initials: S
authors.lastname: Abdelazim
authors.forename: Rasem J
authors.initials: RJ
authors.lastname: Fattah
authors.forename: Shihui
authors.initials: S
authors.lastname: Liu
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Leppla
mesh_terms: D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D001408:Bacillus anthracis / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D045744:Cell Line, Tumor; D002852:Chromatography, Ion Exchange; D004167:Diphtheria Toxin / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D000922:Immunotoxins / Q000302:isolation & purification* / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D016211:Transforming Growth Factor alpha / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D004167:Diphtheria Toxin; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D016211:Transforming Growth Factor alpha; C030325:anthrax toxin