J Bacteriol Virol.  2010 Mar;40(1):29-37. 10.4167/jbv.2010.40.1.29.

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

• Kim YJ
• Baek JH
• Kim JH
• Kim BS
• Rhie GE
• Yoo CK
• Shin NR

• KMID: 1456251
• DOI: http://doi.org/10.4167/jbv.2010.40.1.29

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.

Keyword

Botulinum neurotoxin type A; SNAP-25; Endopeptidase assay

MeSH Terms

Animals
Biological Assay
Blotting, Western
Glutathione Transferase
Immune Sera
Lethal Dose 50
Mice
Neurotransmitter Agents
Peptides
Glutathione Transferase
Immune Sera
Neurotransmitter Agents
Peptides

Figure

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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.

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  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.

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  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.

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  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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