Cloning, expression, and one-step purification of the minimal essential domain of the light chain of botulinum neurotoxin type A

A truncated but functional form of the botulinum neurotoxin A light chain ( Tyr 9-Leu 415) has been cloned into the three bacterial expression vectors, PET 28, pET 30, and PGEX-2T, and produced as fusion proteins. This 406-ami no-acid light chain was expressed with 1 six-histidine tag (LC-pET28), 2 si x histidine tags and a S-tag (LC-pET30), or a six-histidine tag and a gluta thione S-transferase tag (LC-pGEX-2T). The three fusion proteins have been overexpressed in Escherichia coli, purified in a soluble form, and tested f or protease activity. All three recombinant proteins were found to have sim ilar enzymatic activity, comparable to the light chain purified from the wh ole toxin, The LC-pET30 protein was the most soluble and stable of the thre e fusion proteins, and it could be purified using a one-step affinity chrom atography protocol. The purified protein was determined to be 98% pure as a ssessed by SDS-polyacrylamide gel. This protein has been crystallized and i nitial X-ray data show that the crystals diffract to 1.8 Angstrom. (C) 2000 Academic Press.