• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to cleave the fluorogenic peptide substrate pERTKR-AMC (Catalog # ). The specific activity is >750 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  E. coli-derived

  MHHHHHH	NS2b (Ser1423-Lys1470) Accession # ABD85079	GGGGSGGGG	West Nile Virus NS3 (Gly1506-Leu1689) Accession # ABD85079
  		GGGGSGGGG	West Nile Virus NS3 (Gly1506-Leu1689) Accession # ABD85079
  N-terminus			C-terminus

• Accession #

• N-terminal Sequence    
  Analysis

  Met and Gly

• Predicted Molecular Mass

  27 kDa & 20 kDa

• SDS-PAGE

  32 kDa and 22 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM Tris, 30% (v/v) Glycerol, pH 9.5

• Recombinant Viral wnvNS3 Protease (Catalog # 2907-SE)

• Substrate: L-PYROGlu-Arg-Thr-Lys-Arg-AMC (pERTKR-AMC) (Catalog # )

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

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

1. Dilute rwnvNS3 Protease to 1 ng/µL in Assay Buffer.

2. Dilute Substrate to 40 µM in Assay Buffer.

3. In a plate load 50 µL of 1 ng/µL rwnvNS3 Protease, and start the reaction by adding 50 µL of 40 µM Substrate to wells. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 40 µM Substrate.

4. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.

5. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard 7-Amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A-9891).

Per Well:

• rwnvNS3 Protease: 0.05 µg

• Substrate: 20 µM

Background: wnvNS3 Protease

Infection of mosquito-borne West Nile Virus can cause severe neurological disease and can be epidemic. Two non-structural proteins, NS3 and NS2b, play an essential role in viral replication and are therefore a potential target for treatment and prevention of West Nile Virus disease. NS3 consists of a trypsin-like serine protease with a catalytic triad (His51, Asp75, Ser135) and a putative helicase. Requiring NS2b as the co‑factor, NS3 protease processes viral polyprotein precursor (1, 2). The purified recombinant protein consists of three forms: the full-length fusion protein, the N-terminal NS2b, and the C-terminal NS3 with the G₄SG₄ linker. NS3 protease has a relatively narrow substrate specificity that prefers Arg in P1 and Lys in P2. The purified recombinant protein has autocatalytic activity that can lead to protein degradation. It is therefore important to store the sample below -20 °C and to keep on ice while working with the sample.

• References:

1. Nall, T.A. et al. (2004) J. Biol. Chem. 279:48535.

2. Chappell, K.J. et al. (2005) J. Biol. Chem. 274:2896.

• Long Name:

  West Nile Virus NS3 Protease

• Entrez Gene IDs:

  912267 (Viral)

• Alternate Names:

  wnvNS3 Protease