Study on the protease activity of the recombinant light chain of clostridium botulinum type A and type E

Abstract

The light chain fragment of botulinum neurotoxin, serotype A and serotype E, has been cloned to pET21-a using PCR amplification from chromosomal DNA and successfully expressed in E. coli strain. The expressed recombinant light chains were purified using His-bind resin which can bind to histidine tagged sequence conjugated expressed light chain. The zinc-endopeptidase activity and substrate specificity for SNAP 25 of constructed recombinant light chain of BoNT/A and BoNT/E was characterized. The cleavage activity was efficiently antagonized by chelating agent, ethylenediamine tetraacetic acid (EDTA). Distribution of SNAP 25, a target protein of botulinum type A and type E, in neuronal tissues and different nonneuronal tissues was studied using immuno blot analysis. The SNAP 25 was strongly expressed in brain, but little expression was observed in other tissues. In contrast, the SNAP 23, a homolog of SNAP 25, was not expressed in the brain, but strongly expressed in almost all other tissues examined. The results were supported by RT-PCR technique using SNAP 23 primers. In the conclusion, SNAP 23 may play a critical role in the process of membrane fusion in the constitutives pathway instead of SNAP 25. To develop the vaccine against botulinum toxin, Balb/C mice were vaccinated with the cell lysate expressing the light chain of botulinum type A, type B, and type E. The vaccination with crude recombinant L chain led to protection against a dose of up to $10^4$ $LD_50$ of type B botulinum toxin and $10^5$ $LD_50$ of the type A botulinum and $10^4$ $LD_50$ of type E botulinum. From this result, in the vaccination, the purified light chain might be useful for antigen.