Recombinant Expression and Purification of the Botulinum Neurotoxin Type A Translocation Domain

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 thr...

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Bibliographic Details
Published in:Protein expression and purification 1997-11, Vol.11 (2), p.195-200
Main Authors: Lacy, D.Borden, Stevens, Raymond C.
Format: Article
Language:English
Subjects:
Biological Transport
Botulinum Toxins, Type A - genetics
Botulinum Toxins, Type A - isolation & purification
Circular Dichroism
Cloning, Molecular
Clostridium botulinum
Crystallization
Escherichia coli
Escherichia coli - genetics
Neuromuscular Agents - isolation & purification
Peptide Fragments - genetics
Peptide Fragments - isolation & purification
Recombinant Proteins - isolation & purification
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Summary: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 recombinantEscherichia coliexpression 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.
ISSN:1046-5928
1096-0279
DOI:10.1006/prep.1997.0772