title: Recombinant expression and purification of the botulinum neurotoxin type A translocation domain.
abstract: Botulinum neurotoxin type A in its fully activated form exists as a dichain protein consisting of a 50-kDa light chain and a 100-kDa heavy chain linked by a disulfide bond (B. R. DasGupta and H. Sugiyama, Biochem. Biophys. Res. Commun. 48, 108-112, 1972). The protein can be further subdivided into three functional domains: a catalytic domain corresponding to the light chain, a translocation domain associated with the N-terminal half of the heavy chain, and a binding domain as the C-terminal half. To facilitate further structural and functional studies on the mechanism of toxin translocation, we report here the recombinant Escherichia coli expression and purification of the isolated translocation domain with a yield of 1 mg pure protein per 1 g cell paste. Circular dichroism, enzyme-linked immunosorbent assays, and preliminary crystallization experiments verify proper protein folding. This reagent should serve as a key tool in elucidating the mechanism of translocation and in determining how the catalytic domain, a large 50-kDa metalloprotease, is delivered to the cytosol.
authors.affiliation: Department of Chemistry, University of California, Berkeley, California 94720, USA.
authors.forename: D B
authors.initials: DB
authors.lastname: Lacy
authors.forename: R C
authors.initials: RC
authors.lastname: Stevens
mesh_terms: D001692:Biological Transport; D019274:Botulinum Toxins, Type A / Q000235:genetics / Q000302:isolation & purification*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics; D009465:Neuromuscular Agents / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification
chemical_list: D009465:Neuromuscular Agents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019274:Botulinum Toxins, Type A