• Purity

  >90%, 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 4 ng, as measured under the decribed conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  E. coli-derived Thr2-Ser428, with an N-terminal Met and 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met

• Predicted Molecular Mass

  50 kDa

• SDS-PAGE

  41-45 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM HEPES, pH 6.5

• Recombinant C. botulinum BoNT‑D Light Chain (rBoNT/D-LC) (Catalog # 6037-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 rBoNT/D-LC to 0.4 µg/mL in Assay Buffer.

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

4. Incubate reaction vials at room temperature for 1 hour. Incubate one control at room temperature 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.

Per Lane:

• rBoNT/D-LC: 4 ng

• Substrate: 1 µg

Background: BoNT-D Light Chain

Botulinum Neurotoxin Type D 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 that are activated by proteolytic cleavage to create the light and heavy chains that are linked by a disulfide bond. The 50 kDa light chain contains the metalloprotease domain whereas the 100 kDa heavy chain contains a receptor binding domain and a domain required for translocation across the cell membrane (3). BoNTs are the most toxic protein toxins known for humans. As zinc proteases, they cleave SNARE proteins to elicit flaccid paralysis in botulism poisoning. Cleavage of the SNARE proteins results in the blocking of acetylcholine release at the neuromuscular junction (2‑4).   E. coli expressed recombinant light chains are active proteases.  In the absence of heavy chains, however, they lack toxicity because they cannot enter into host cells.

• References:

1. Campbell, K.D. et al. (1993) J. Clin. Microbiol. 31:2255.

2. Montecucco, C. and S. Giampietro (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.

• Long Name:

  Botulinum Neurotoxin Type D Light Chain

• Alternate Names:

  BoNTD Light Chain; BoNT-D Light Chain