Recombinant M. viridifaciens Neuraminidase Protein, CF

• Reactivity: Mv
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant M. viridifaciens Neuraminidase Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a fluorogenic substrate, 2’-(4-Methylumbelliferyl)-alpha -D-N-acetylneuraminic acid. The specific activity is >25,000 pmol/min/µg, as measured under the described conditions.
• Source: E. coli-derived m. viridifaciens Bacterial Neuraminidase protein
  Pro42-Gly403, with an N-terminal Met and 6-His tag
  Accession # BAA00852
• Accession #: BAA00852
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, 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

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 40 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: 42 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 and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 50 mM Sodium Acetate, 150 mM NaCl, pH 4.5
  • Recombinant M. viridifaciens Neuraminidase (rMvNA) (Catalog # 5084-NM)
  • Substrate: 2’-(4-Methylumbelliferyl)-alpha -D-N-acetylneuraminic acid (Sigma, Catalog # M8639), 10 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 rMvNA to 0.2 ng/µL in Assay Buffer.
  2. Dilute Substrate to 400 µM with Assay Buffer.
  3. Load into plate 50 µL of 0.2 ng/µL rMvNA and start the reaction by adding 50 µL of 400 µM Substrate. Include a Substrate Blank containing 50 µL of Substrate and 50 µL of Assay Buffer.
  4. Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively, in kinetic mode for 5 minutes.
  5. Calculate specific activity:

  Specific Activity (pmoles/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmole/RFU)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank

  **Derived using calibration standard 4-Methylumbelliferone (Sigma, Catalog # M1381).

  • rMvNA: 0.010 µg
  • Substrate: 200 µM

Notes

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

Alternate Names for Recombinant M. viridifaciens Neuraminidase Protein, CF

• Bacterial Neuraminidase

Background

Neuraminidase is the common name for N-acetyl-neuraminyl hydrolase (Sialidase). Micromonospora viridifaciens Neuraminidase catalyzes the hydrolysis of alpha 2-3 and alpha 2-6 and alpha 2-8 linked N-acetyl-neuraminic acid residues from glycoproteins and glycolipids (1). The architecture of the full-length protein includes a canonical neuraminidase enzymatic domain, a linker domain and a C-terminal galactose binding domain (2). The full-length protein contains 647 amino acids and has a molecular weight of 68 kDa. It is easily degraded to 52 kDa and 41 kDa fragments (3). The expressed enzyme only contains the enzymatic domain.

1. Aisaka, K. & Uwajima, T. (1987) FEBS Microbiol. Lett. 44:289.
2. Gaskell, A. et al. (1995) Structure 3:1197.
3. Sakurada, K. et al. (1992) J. Bacteriol. 174:6896.

Bioinformatics

• Uniprot:
  • BAA00852()