Recombinant Tetanus Neurotoxin Type Light Chain Protein, CF

• Reactivity: Ba-Ct
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Tetanus Neurotoxin Type Light Chain Protein, CF Summary

• Details of Functionality: 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.
• Source: E. coli-derived c. tetani TeNT Light Chain protein
  Pro2-Gly430, with an N-terminal Met and 6-His tag
• Accession #: P04958
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: tetX
• Purity: >85%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 50 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 43-50 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Tween® 20.
• Purity: >85%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • 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 # 7375-SV)
  • 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

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd. Tween is a registered trademark of ICI Americas.

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Tetanus Neurotoxin Type Light Chain Protein, CF

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

Background

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.

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.

Publications for TeNT Light Chain/Tetanus Toxin (6535-ZN)(1)

Publication using 6535-ZN

• Albarran, E;Sun, Y;Liu, Y;Raju, K;Dong, A;Li, Y;Wang, S;S�dhof, TC;Ding, JB; Postsynaptic synucleins mediate endocannabinoid signaling Nature neuroscience 2023-05-29 [PMID: 37248337] (Bioassay, N/A, Mouse)

Bioinformatics

• Gene Symbol: tetX
• Uniprot:
  • P04958(C. tetani)