Recombinant West Nile Virus NS3 Protease Protein, CF

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

Recombinant West Nile Virus NS3 Protease Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate pERTKR-AMC (Catalog # ES013). The specific activity is >750 pmol/min/µg, as measured under the described conditions.
• Source: E. coli-derived viral wnvNS3 Protease protein
  

  MHHHHHH	NS2b (Ser1423-Lys1470) (Ser1435Thr) Accession # YP_001527877.1	GGGGSGGGG	West Nile Virus NS3 (Gly1506-Leu1689) Accession # YP_001527877.1
  		GGGGSGGGG	West Nile Virus NS3 (Gly1506-Leu1689) Accession # YP_001527877.1
  N-terminus			C-terminus

• Accession #: YP_001527877.1
• N-terminal Sequence: Met and Gly
• Protein/Peptide Type: Recombinant Enzymes
• Gene: flavivirus polyprotein gene
• 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: 27 kDa & 20 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: 32 kDa and 18-22 kDa, under 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 reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Reconstitution Instructions: Reconstitute at 200 μg/mL in sterile, deionized water.
• Assay Procedure:
  • 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 # ES013)
  • 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:

  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 7-Amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A-9891).

  Per Well:
  • rwnvNS3 Protease: 0.05 µg
  • Substrate: 20 µM

Notes

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

Alternate Names for Recombinant West Nile Virus NS3 Protease Protein, CF

• wnvNS3 Protease

Background

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.

1. Nall, T.A. et al. (2004) J. Biol. Chem. 279:48535.
2. Chappell, K.J. et al. (2005) J. Biol. Chem. 274:2896.

Publications for wnvNS3 Protease (2907-SE)(3)

Publications using 2907-SE

• J Pianka, N Gruba, A Lesner Novel tools to study West Nile virus NS3 protease activity Bioorganic chemistry, 2023-02-15;133(0):106426. 2023-02-15 [PMID: 36801793] (Bioassay, N/A)
• F Uliana, M Vizovišek, L Acquasalie, R Ciuffa, A Fossati, F Frommelt, S Goetze, B Wollscheid, M Gstaiger, V De Filippi, U Auf dem Ke, R Aebersold Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen Nature Communications, 2021-03-16;12(1):1693. 2021-03-16 [PMID: 33727531] (Bioassay, Human)
• AS de Oliveir, PAR Gazolla, AFCDS Oliveira, WL Pereira, LC de S Viol, AFDS Maia, EG Santos, ÍEP da Silva, TAO Mendes, AM da Silva, RS Dias, CC da Silva, MD Polêto, RR Teixeira, SO de Paula Discovery of novel West Nile Virus protease inhibitor based on isobenzonafuranone and triazolic derivatives of eugenol and indan-1,3-dione scaffolds PLoS ONE, 2019-09-26;14(9):e0223017. 2019-09-26 [PMID: 31557229] (Bioassay, Virus)

Bioinformatics

• Gene Symbol: flavivirus polyprotein gene
• Uniprot:
  • YP_001527877.1()