Recombinant Influenza A Virus H5N1 Neuraminidase Protein, CF 10 UG

• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane

• Endotoxin Level

  <0.10 EU per 1 μg of the protein by the LAL method.  

• Activity

  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. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Ser37-Lys449, with an N-terminal vasodilator-stimulated phosphoprotein tetramerization domain and a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Ser (tetramerization domain)

• Predicted Molecular Mass

  51 kDa

• SDS-PAGE

  58-65 kDa, reducing conditions

Assay Procedure

Materials

• Activation Buffer: 50 mM MES, 500 mM NaCl, 30 mM CaCl₂, pH 6.5

• Assay Buffer: 50 mM MES, 500 mM NaCl, 5 mM CaCl₂, pH 6.5

• Recombinant Influenza A Virus H5N1 Neuraminidase (rvH5N1 Neuraminidase) (Catalog # 7597-NM)

• Substrate: 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: Gemini EM by Molecular Devices) or equivalent

1. Dilute rvH5N1 Neuraminidase to 100 μg/mL in Activation Buffer.

2. Incubate for 24 hours at 37 °C to activate.

3. Dilute activated rvH5N1 Neuraminidase to 0.5 ng/μL in Assay Buffer.

4. Dilute Substrate to 400 µM in Assay Buffer.

5. Load into a black well plate 50 µL of the 0.5 ng/μL rvH5N1 Neuraminidase and start the reaction by adding 50 µL of 400 µM Substrate. Include a Substrate Blank containing Assay Buffer in place of rvH5N1 Neuraminidase.

6. Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively, in kinetic mode for 5 minutes.

7. 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:

• rvH5N1 Neuraminidase: 0.025 µg

• Substrate: 200 µM

Background: Viral Neuraminidase

Neuraminidase (NA) and hemagglutinin (HA) are the two predominant membrane glycoproteins found on the surface of an influenza virus particle. They are essential for the infectious cycle of the virus. HA recognizes and binds to the sialic acid on the host cell membrane to initiate a viral infection. NA cleaves the sialic acid at the end of the cycle, allowing the progeny virus to leave the host and initiate the next round of infection (1). In the early stage of an infection, NA may also assist in viral penetration of the mucus layer in the airway of a host. Nine subtypes of NA (N1 to N9) have been identified, all of which are believed to be tetrameric and share a basic structure consisting of a globular head, a thin stalk region, and a small hydrophobic region that anchors the protein in the virus membrane (2). Glycosylation has also found been important for the stability and activity of these enzymes (3, 4). According to a recent structure determination (5), there are two genetically distinct groups of neuraminidases from influenza type A viruses, with the N1 and N2 neuraminidases representing the two groups. Due to their critical role in the infectious cycle of a virus, influenza viral neuraminidases are frequently used as targets for drug design. In fact, both Tamiflu and Relenza, anti-influenza drugs, are neuraminidase inhibitors. Our recombinant H5N1 neuraminidase is based on the avian virus isolated from the 2004 outbreaks of the H5N1 virus in Vietnam and Thailand (6). H5N1 avian virus is one of the most lethal viruses in history (7, 8) with an accumulative death rate of 59% from 2003 to 2012 according to the World Health Organization (9). To produce active recombinant enzyme, a tetramerization domain from the vasodilator-stimulated phosphoprotein (10) was inserted at the N‑terminus to assist in oligomerization of the protein. We found that the activity of the recombinant H5N1 neuraminidase is activated by Ca  ²⁺ and inactivated by Zn  ²⁺, Cu  ²⁺ and Fe  ²⁺.

• References:

1. Palese, P. & Compans, R. W. (1976) J. Gen. Virol. 33:159.

2. Colman, P. M. et al. (1983) Nature 303:41.

3. Wu, Z.L. et al. (2009) Biochem. Biophys. Res. Commun. 379:749.

4. Sun, S. et al. (2012) PLoS one 7:e32119.

5. Russell, R.J. et al. (2006) Nature 443:45.

6. Govorkova, E.A. et al. (2005) J. Virol. 79:2191.

7. Ducatez, M.F. et al. (2006) Nature 442:37.

8. Peiris, J.S. et al. (2007) Clin. Microbiol. Rev. 20:243.

9. 
   

10. Kuhnel, K. et al. (2004) Proc. Natl. Acad. Sci. U. S. A. 101:17027.

• Alternate Names:

  NANH; Viral Neuraminidase