• Purity

  >85%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie? Blue stain at 5 μg per lane

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by the cleavage of the substrate GFPuv/SNAP/VAMP in a gel-shift assay. >50% of 1 μg substrate is cleaved by 40 ng, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  E. coli-derived Pro2-Gly430, with an N-terminal Met and 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met

• Predicted Molecular Mass

  50 kDa

• SDS-PAGE

  43-50 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM HEPES, 0.05% (v/v) Tween? 20, pH 7.5

• Recombinant C. tetani TeNT Light Chain (rTeNT-LC) (Catalog # 6535-ZN)

• Substrate: Recombinant GFP/SNAP25B/VAMP-2 (Catalog # )

• SDS-PAGE and silver stain reagents or equivalent or Western blot with appropriate antibodies

1. Dilute Substrate to 100 μg/mL in Assay Buffer.

2. Dilute rTeNT-LC to 4 μg/mL in Assay Buffer.

3. Combine equal volumes of diluted Substrate with diluted rTeNT-LC. Prepare two controls by combining equal volumes of diluted Substrate with Assay Buffer.

4. Incubate reaction vials at 37 °C for 1 hour.  Incubate one control at 37 °C and the other at -20 °C for 1 hour.

5. After incubation, combine reaction mixtures and controls with reducing SDS-PAGE sample buffer at a 1:1 (reaction mixture:sample buffer) ratio (v/v) to stop reactions.

6. Analyze the cleavage products by SDS-PAGE (Load 40 μL of the mixture from step 5 per lane, 1 μg Substrate per lane) followed by silver staining and/or Western blot.

• rTeNT-LC:  40 ng

• Substrate:  1 μg

Background: TeNT Light Chain

Tetanus toxin (TeNT) is a potent neurotoxin produced by   Clostridium tetani. TeNT is similar in structure and function to the botulinum toxins produced by other Clostridium species (1, 2). TeNT is among the most toxic protein toxins known for humans. TeNT is synthesized as an inactive single chain precursor that undergoes proteolytic cleavage to create light and heavy chains that are linked by a disulfide bond. The 50 kDa light chain contains a metalloprotease domain whereas the 100 kDa heavy chain contains a receptor binding domain and a domain required for translocation across the cell membrane (3). Once inside a neuronal cell the zinc metalloprotease domain is able to cleave synaptobrevin to cause motor neuron disinhibition, resulting in the spastic paralysis characteristic of tetanus (4). The   E. coli expressed recombinant TeNT light chain is an active protease. In the absence of heavy chains, however, it lacks toxicity because it cannot enter into host cells.

• References:

1. Eisel, U. et al. (1986) EMBO J. 5:2495.

2. Montecucco, C. and S. Giampietro (1993) Trends Biochem. Sci. 18:324.

3. Schiavo, G. et al. (1992) Nature 359:832.

4. Schiavo, G. et al. (1992) EMBO J. 11:3577.

• Long Name:

  Tetanus Neurotoxin Type Light Chain

• Entrez Gene IDs:

  1061100 (C. tetani)

• Alternate Names:

  TeNT Light Chain; TeNT-LC; Tentoxylysin; Tetanus toxin chain H; Tetanus toxin chain L; Tetanus toxin heavy chain; Tetanus toxin light chain; tetX