Cloning, high level expression, purification, and crystallization of the full length Clostridium botulinum neurotoxin type E light chain

The catalytic activity of the highly potent botulinum neurotoxins are confined to their N-terminal light chains (similar to50 kDa). A full-length light chain for the type E neurotoxin with a C-terminal 6x His-tag, BoNT/E-LC, has been cloned in a pET-9c vector and over-expressed in BL21 (DE3) cells. BoNT/E-LC was purified to homogeneity by affinity chromatography oil Ni-NTA agarose followed by exclusion chromatography using a Superdex-75 sizing column. The purified protein has very good solubility and call be stored stably at -20 degreesC; however, it seems to undergo auto-proteolysis when stored at temperature greater than or equal to4-10 degreesC. BoNT/E-LC is active on its natural substrate, the synaptosomal associated 25 kDa protein, SNAP-25, indicating that it retains a native-like conformation and therefore can be considered as a useful tool in studying the structure/function of the catalytic light chain. Recombinant BoNT/E-LC has been crystallized under five different conditions and at various pHs. Crystals diffract to better than 2.1 Angstrom. (C) 2003 Elsevier Inc. All rights reserved.