Recombinant Influenza A Virus H1N1 Neuraminidase Protein, CF

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

Recombinant Influenza A Virus H1N1 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 >2,500 pmol/min/µg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived influenza a virus h1n1 Viral Neuraminidase protein
  Ser37-Lys469, with an N-terminal 6-His tag
• Accession #: AAF77036
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >80%, 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: 48 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: 57 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 Glycerol.
• Purity: >80%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 5 mM CaCl₂, 200 mM NaCl, pH 7.5
  • Recombinant Influenza A Virus H1N1 Neuraminidase (rH1N1 Neuraminidase) (Catalog # 4858-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 rH1N1 Neuraminidase to 0.05 ng/µL in Assay Buffer.
  2. Dilute Substrate to 400 µM in Assay Buffer.
  3. Load 50 µL of 0.05 ng/µL rH1N1 Neuraminidase into the plate, and start the reaction by adding 50 µL of 400 µM Substrate. As a Substrate Blank load 50 µL of Assay Buffer and 50 µL of Substrate.
  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 (pmol/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)) / (amount of enzyme (µg))

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

  Per Well:
  • rH1N1 Neuraminidase: 0.0025 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 Influenza A Virus H1N1 Neuraminidase Protein, CF

• NANH
• Sialidase
• Viral Neuraminidase

Background

Neuraminidase (NA) and hemagglutinin (HA) are major membrane glycoproteins found on the surface of influenza virus. HA is a lectin that binds sialic acid on host cell membrane. NA is a sialic acid hydrolase that specifically clips off terminally located sialic acid on host cell surface. The two proteins are essential for the infectious cycle of the influenza virus. During initial infection, an influenza virus will hold onto an epithelial cell through HA-sialic acid interaction. At the end of an infectious cycle, the NA will cleave the sialic acid on the host cell membrane, releasing the formed viral particle from the HA-sialic acid bondage (1). The neuraminidase activity is also thought to help the virus penetrate mucus. Nine subtypes of NA have been identified, all of which are tetrameric and share a common structure consisting of a globular head, a thin stalk region, and a small hydrophobic region that anchors the protein in the virus membrane (2). The purified rvH1N1NA consists of amino acid residues 37 to 469 as deduced from the 1918 Spanish flu virus NA (A/Bervig_Mission/1/18) (3). It has a distinct N-glycan profile and is resistant to trypsin digestion (4).

1. Palese, P. & Compans, R. W. (1976) J. Gen. Virol. 33:159.
2. Colman, P. M. et al. (1983) Nature 303:41.
3. Reid, A. (2000) Proc. Natl. Acad. Sci. USA 97:6785.
4. Wu, Z.L. et al. (2009) Biochem. Biophys. Res. Comm. 379:749.

Publications for Neuraminidase (4858-NM)(2)

Publications using 4858-NM

• Watson DC, Leclerc S, Wakarchuk WW, Young NM Enzymatic synthesis and properties of glycoconjugates with legionaminic acid as a replacement for neuraminic acid. Glycobiology, 2010-10-25;21(1):99-108. 2010-10-25 [PMID: 20978010] (Enzyme Assay)
• Wu ZL, Ethen C, Hickey GE, Jiang W Active 1918 pandemic flu viral neuraminidase has distinct N-glycan profile and is resistant to trypsin digestion. Biochem. Biophys. Res. Commun., 2009-01-06;379(3):749-53. 2009-01-06 [PMID: 19133226] (Enzyme Assay, Virus)

Bioinformatics

• Uniprot:
  • AAF77036(Viral)