• Purity

  >90%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.1 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to cleave the fluorogenic peptide substrate, SNAPtide. The specific activity is >1 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  E. coli-derived Met1-Phe425, with an N-terminal Met and 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met

• Predicted Molecular Mass

  50 kDa

• SDS-PAGE

  52 kDa, reducing conditions

Assay Procedure

Materials

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

• Recombinant C. botulinum BoNT?A Light Chain (rBoNT/A-LC) (Catalog # 4489-ZN)

• Substrate: SNAPtide (FITC/DABCYL) (List Biological Laboratories, Inc., Catalog # 521), 2 mM stock in DMSO

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: Spectra Max Gemini EM by Molecular Devices) or equivalent

1. Dilute rBoNT/A-LC to 10 μg/mL in Assay Buffer.

2. Dilute Substrate to 10 μM in Assay Buffer.

3. Load into a black well plate 50 μL of 10 μg/mL of rBoNT/A-LC and start the reaction by adding 50 μL of 10 μM Substrate. As a Substrate Blank combine 50 μL of 10 μM Substrate and 50 μL of Assay Buffer.

4. Read at excitation and emission wavelengths of 490 nm and 523 nm (top read), respectively, in kinetic mode for 5 minutes.

5. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard Unquenched Calibration Peptide (List Biological Laboratories, Catalog # 528).

Per Well:

• rBoNT/A-LC: 0.50 μg

• Substrate: 5 μM

Background: BoNT-A Light Chain

Botulinum neurotoxin type A 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. However, they are not toxic because they cannot enter into host cells in the absence of the heavy chains.

• References:

1. Willems, A. et al. (1993) Res. Microbiol. 144:547.

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.

• Long Name:

  Botulinum Neurotoxin Type A Light Chain

• Entrez Gene IDs:

  5185061 (C. botulinum)

• Alternate Names:

  BoNTA Light Chain; BoNT-A Light Chain