Detection of Botulinum Neurotoxin Type A by In Vitro Bioassay Based on Endopeptidase Activity.
- Author:
Yun Jeong KIM
1
;
Joung Hee BAEK
;
Jeong Hee KIM
;
Bong Su KIM
;
Gi eun RHIE
;
Cheon Kwon YOO
;
Na Ri SHIN
Author Information
- Publication Type:In Vitro ; Original Article
- Keywords: Botulinum neurotoxin type A; SNAP-25; Endopeptidase assay
- MeSH: Animals; Biological Assay; Blotting, Western; Glutathione Transferase; Immune Sera; Lethal Dose 50; Mice; Neurotransmitter Agents; Peptides
- From:Journal of Bacteriology and Virology 2010;40(1):29-37
- CountryRepublic of Korea
- Language:Korean
- Abstract: Botulinum neurotoxin type A (BoNT/A) is a metalloprotease that cleaves SNAP-25 (synaptosome-associated protein of 25 kDa), a specific cellular protein essential for neurotransmitter release. As well as mouse bioassay to detect BoNT/A, various assay methods based on its endopeptidase activity have been developed. In this study, we tried to develop a BoNT/A assay system using recombinant SNAP-25 with glutathione S-transferase (GST) tags at both termini as substrate. The recombinant GST-SNAP-25-GST with 70 kDa was expressed and purified in E. coli and synthesized N-terminal 50 kDa and C-terminal 25 kDa fragment after cleavage at the Gln(197)-Arg(198) bond by BoNT/A. To detect both fragments, we obtained rabbit antisera against peptides corresponding to the cleaved ends of each fragment. In the western blotting, the N-terminal fragment was detected by the antibody specifically recognizing the newly exposed C-terminus (corresponding to amino acid residue 191-197). This assay system was able to detect until 3.125 ng of BoNT/A, which corresponded to about 90 fold LD50 in mice. These results suggest that the in vitro endopeptidase assay developed in this study would replace others to detect BoNT/A.
