Recombinant Botulinum Neurotoxin Type E Light Chain, CF

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Summary
Reactivity CbSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

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Recombinant Botulinum Neurotoxin Type E Light Chain, CF Summary

Details of Functionality
Measured by its ability to cleave the fluorogenic peptide substrate, SNAPEtide. The specific activity is >85 pmol/min/μg, as measured under the described conditions.
Source
E. coli-derived c. botulinum BoNT-E Light Chain protein
Pro2-Ile403, with an N-terminal Met and 6-His tag
Accession #
N-terminal Sequence
Met
Protein/Peptide Type
Recombinant Enzymes
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Theoretical MW
46 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 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, Glycerol, Brij-30 and DTT.
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain
Assay Procedure
  • Assay Buffer: 50 mM HEPES, 0.1% (v/v) Tween® 20, pH 8.0
  • Recombinant C. botulinum BoNT-E Light Chain (rBoNT-E LC) (Catalog # 5424-ZN)
  • Substrate: SNAPEtide (o-Abz/Dnp) (List Biological Laboratories, Inc., Catalog # 550), 2 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rBoNT-E LC to 10 µg/mL in Assay Buffer.
  2. Dilute Substrate to 40 µM in Assay Buffer.
  3. Load into a black well plate 50 µL of 10 µg/mL of rBoNT-E LC, and start the reaction by adding 50 µL of 40 µM Substrate. Include a Substrate Blank containing Assay Buffer and 40 µM Substrate without rBoNT-E LC.
  4. Read at excitation and emission wavelengths of 320 nm and 410 nm (top read), respectively, in kinetic mode for 5 minutes.
  5. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)
amount of enzyme (µg)

     *Adjusted for Substrate Blank

     **Derived using calibration standard Abz-Gly (Bachem, Catalog # E-2920).

Per Well:
  • rBoNT-E LC: 0.5 µg
  • Substrate: 20 µM

Notes

Tween is a registered trademark of ICA Americas.

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

Alternate Names for Recombinant Botulinum Neurotoxin Type E Light Chain, CF

  • BoNTE Light Chain
  • BoNT-E Light Chain

Background

Botulinum Neurotoxin Type E is one of the seven serotypes of Botulinum Neurotoxins (BoNTs) produced by various strains of Clostridium botulinum (1, 2). BoNTs are synthesized as inactive single chain protein precursors and activated by proteolytic cleavage to generate disulfide-linked two-chain proteins. The 50 kDa light chain contains the catalytic domain, whereas the 100 kDa heavy chain contains an internal translocation domain and a receptor binding domain (3). BoNTs are the most potent protein toxins for humans. As zinc proteases, they cleave SNARE proteins to elicit flaccid paralysis in botulism by blocking acetylcholine release at the neuromuscular junction (2‑4). E. coli-expressed recombinant light chains are active proteases. In the absence of the heavy chains, however, they lack toxicity because they cannot enter into host cells.

  1. Campbell K.D. et al. (1993) J. Clin. Microbiol. 31:2255.
  2. Montecucco, C. and Giampietro, S. (1993) Trends. Biochem. Sci. 18:324.
  3. Turton, K. et al. (2002) Trends. Biochem. Sci. 27:552.
  4. Schiavo, G. et al. (2000) Physiol. Rev. 80:717.

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