Recombinant Influenza A Virus H5N1 Neuraminidase Protein, CF

Catalog #: 7597-NM
3 Citations Datasheet / COA / SDS

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7597-NM-010

R&D Systems Recombinant Proteins and Enzymes

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All Viral Neuraminidase Proteins and Enzymes

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Influenza A Virus H5N1 Neuraminidase Protein, CF Summary

Product Specifications

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.

Source

Mouse myeloma cell line, NS0-derived influenza a virus h5n1 Viral Neuraminidase protein
Ser37-Lys449, with an N-terminal vasodilator-stimulated phosphoprotein tetramerization domain and a C-terminal 6-His tag

Accession #

Q6DPL2

N-terminal Sequence
Analysis

Ser (tetramerization domain)

Predicted Molecular Mass

51 kDa

SDS-PAGE

58-65 kDa, reducing conditions

Product Datasheets

7597-NM

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

7597-NM

Formulation	Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
Shipping	The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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.

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

Dilute rvH5N1 Neuraminidase to 100 μg/mL in Activation Buffer.
Incubate for 24 hours at 37 °C to activate.
Dilute activated rvH5N1 Neuraminidase to 0.5 ng/μL in Assay Buffer.
Dilute Substrate to 400 µM in Assay Buffer.
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.
Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively, in kinetic mode for 5 minutes.
Calculate specific activity:

Specific Activity (pmol/min/µg) =
Adjusted V_max* (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

Reconstitution Calculator

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 Ca²⁺ and inactivated by Zn²⁺, Cu²⁺ and Fe²⁺.

References

Palese, P. & Compans, R. W. (1976) J. Gen. Virol. 33:159.
Colman, P. M. et al. (1983) Nature 303:41.
Wu, Z.L. et al. (2009) Biochem. Biophys. Res. Commun. 379:749.
Sun, S. et al. (2012) PLoS one 7:e32119.
Russell, R.J. et al. (2006) Nature 443:45.
Govorkova, E.A. et al. (2005) J. Virol. 79:2191.
Ducatez, M.F. et al. (2006) Nature 442:37.
Peiris, J.S. et al. (2007) Clin. Microbiol. Rev. 20:243.
http://www.who.int/influenza/human_animal_interface/EN_GIP_20120405NCumulativeNumberH5N1cases.pdf
Kuhnel, K. et al. (2004) Proc. Natl. Acad. Sci. U. S. A. 101:17027.

Alternate Names

NANH; Sialidase; Viral Neuraminidase

Product Specific Notices

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

Related Research Areas

Citations for Recombinant Influenza A Virus H5N1 Neuraminidase Protein, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

3 Citations: Showing 1 - 3
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