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KMID : 0921620100400010029
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Journal of Bacteriology and Virology
2010 Volume.40 No. 1 p.29 ~ p.37
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Detection of Botulinum Neurotoxin Type A by In Vitro Bioassay Based on Endopeptidase Activity
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Kim Yoon-Jung
Baek Joung-Hee
Kim Jeong-Hee
Kim Bong-Su
Rhie Gi-Eun
Yoo Cheon-Kwon
Shin Na-Ri
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Abstract
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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 Gln197-Arg198 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.
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KEYWORD
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Botulinum neurotoxin type A, SNAP-25, Endopeptidase assay
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