Borden Lacy
Last active: 3/24/2020

Recombinant expression and purification of the botulinum neurotoxin type A translocation domain.

Lacy DB, Stevens RC
Protein Expr Purif. 1997 11 (2): 195-200

PMID: 9367816 · DOI:10.1006/prep.1997.0772

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.

Copyright 1997 Academic Press.

MeSH Terms (9)

Biological Transport Botulinum Toxins, Type A Circular Dichroism Cloning, Molecular Crystallization Escherichia coli Neuromuscular Agents Peptide Fragments Recombinant Proteins

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