Detection of Botulinum Neurotoxin Type A by In Vitro Bioassay Based on Endopeptidase Activity

Yun Jeong Kim; Joung Hee Baek; Jeong-Hee Kim; Bong Su Kim; Gi-eun Rhie; Cheon-Kwon Yoo; Na-Ri Shin

doi:10.4167/jbv.2010.40.1.29

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Kim, Baek, Kim, Kim, Rhie, Yoo, and Shin: Detection of Botulinum Neurotoxin Type A by In Vitro Bioassay Based on Endopeptidase Activity

Original Article

J Bacteriol Virol 2010;40(1):29-37.

Published online: 18 January 2010

DOI: https://doi.org/10.4167/jbv.2010.40.1.29

Detection of Botulinum Neurotoxin Type A by In Vitro Bioassay Based on Endopeptidase Activity

Yun Jeong Kim, Joung Hee Baek, Jeong-Hee Kim, Bong Su Kim, Gi-eun Rhie, Cheon-Kwon Yoo, Na-Ri Shin^∗,^∗∗

Division of High-risk Pathogen Research, Center for Infectious Diseases, National Institute of Health, Seoul, Korea

^∗Corresponding author: Na-Ri Shin. Division of High-risk Pathogen Research, Center for Infectious Diseases, National Institute of Health, 5-Nokbun-dong, Eunpyung-gu, Seoul 122-701, Korea. Phone: +82-2-380-2123, Fax: +82-2-380-1487 e-mail: shinnari1@hanmail.net

^∗∗ This study was financially supported by intramural grant from Control Disease Center, Korea (Project No. 06-BT감염-1).

Revised 10 November 200929 December 2009 Accepted 6 January 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 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.

Keywords: Botulinum neurotoxin type A, SNAP-25, Endopeptidase assay

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Figure 1.

The schematic description of SNAP-25 sequence tagged with glutathione S-transferase (GST) at both termini. The number of amino acid residues corresponds to the sequence from native mouse SNAP-25. The peptide bond site of SNAP-25 cleaved by BoNT/A is between G₁₉₇-A₁₉₈ as indicated.in the SNAP-25. Two black boxes indicate the amino acid sequences of peptides synthesized to produce rabbit antisera.

Figure 2.

The recombinant GST-SNAP-25-GST proteins in the expression and purification steps. (A) Samples from the each step were separated on 10% SDS-PAGE gel and visualized with Coomassie blue staining. Lane 1, protein markers; lane 2, transformed E. coli before IPTG induction; lane 3, transformed E. coli after IPTG induction for 4 h; lane 4, a sonicated bacterial pellet; lane 5, input sample for glutathione affinity; lane 6, flow-through in glutathione affinity; lane 7, GST-SNAP-25-GST purified with glutathione bead. (B) The purity of recombinant GST-SNAP-25-GST was confirmed by SDS-PAGE. Recombinant GST-SNAP-25-GST purified with glutathione bead was buffer-changed by dialysis and concentrated. Lane 1, protein markers; lane 2, the final product of recombinant GST-SNAP-25-GST purified from culture supernatant of transformed E. coli.

Figure 3.

Efficacy measurement of anti-SNAP-25 antisera. The antisera were raised against peptides around newly exposed termini of the SNAP-25 fragments cleaved by BoNT/A from rabbits. (A) ELISA analysis of CRI-R1 and CRI-R2 antisera. These antisera were raised against peptides around newly exposed terminus of N-terminal SNAP-25 fragment by toxin. (B) ELISA analysis of RAT-R1 and RAT-R2 antisera. These antisera were raised against peptides around newly exposed terminus of C-terminal SNAP-25 fragment by toxin. Western blot analysis with CRI-R1 and CRI-R2 (C) and RAT-R1 and RAT-R2 (D) antisera after endopeptidase assay. Forty micromoles of GST-SNAP-25-GST were reacted with 360 μg of BoNT/A in 10 μl for 4 h at 37°C, separated in 10% SDS-PAGE, and analyzed by western blotting with sera. The antisera detected about 50 kDa N-terminal fragment of SNAP-25 cleaved by active BoNT/A. GST (3 μg) was used as a negative control. Lane M, protein markers; lane 1, GST only; lane 2, GST reacted with BoNT/A; lane 3, GST-SNAP-25-GST only; Lane 4, GST-SNAP-25-GST reacted with BoNT/A.

Figure 4.

Detective sensitivity of BoNT/A in the endopeptidase assay using GST-SNAP-25-GST. (A) SDS-PAGE analysis. Forty micromoles of GST-SNAP-25-GST were reacted with various concentrations of BoNT/A in 10 μl for 4 h at 37°C, separated in 10% SDS-PAGE and visualized with Coomassie blue staining to detect 50 kDa N-terminal fragment of SNAP-25 (arrow). (B) Western blot analysis with anti-SNAP-25 antiserum. After the reaction by BoNT/A with the indicated concentrations and separation in SDS-PAGE with the same procedure described in (A), the products of GST-SNAP-25-GST were detected with CRI-R1 antiserum, which detect only 50 kDa N-terminal fragment of SNAP-25 (arrow). The undigested full-length GST-SNAP-25-GST was detected weakly around 75 kDa.

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