RECOMBINANT EXPRESSION AND PURIFICATION OF THE BOTULINUM NEUROTOXIN TYPE-A TRANSLOCATION DOMAIN

Botulinum neurotoxin type A in its fully activated form exists as a di chain protein consisting of a 50-kDa light chain and a 100-kDa heavy c hain linked by a disulfide bond (B. R. DasGupta and H. Sugiyama, Bioch em. Biophys. Res. Commun. 48, 108-112, 1972). The protein can be furth er subdivided into three functional domains: a catalytic domain corres ponding to the light chain, a translocation domain associated with the N-terminal half of the heavy chain, and a binding domain as the C-ter minal 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 transloca tion domain with a yield of 1 mg pure protein per 1 g cell paste. Circ ular dichroism, enzyme-linked immunosorbent assays, and preliminary cr ystallization experiments verify proper protein folding. This reagent should serve as a key tool in elucidating the mechanism of translocati on and in determining how the catalytic domain, a large 50-kDa metallo protease,is delivered to the cytosol. (C) 1997 Academic Press.