Recombinant Human Vanin-1/VNN1 Protein, CF

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

Recombinant Human Vanin-1/VNN1 Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze pantetheine to pantothenate and cysteamine. The specific activity is >1500 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Vanin-1/VNN1 protein
  Gln22-Ser490, with a C-terminal 6-His tag
• Accession #: O95497
• N-terminal Sequence: No results obtained: Gln22 predicted
• Protein/Peptide Type: Recombinant Enzymes
• Gene: VNN1
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <0.01 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 53 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: 60-80 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in HEPES, NaCl, DTT and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 50 mM HEPES, 2 mM DTT, 1% Brij-35 (w/v), pH 7.0
  • Recombinant Human Vanin-1/VNN1 (rhVNN1) (Catalog # 7999-AH)
  • Substrate: Pantethine (Sigma, Catalog # P2125) 50 mM stock in deionized water
  • o-pthalaldehyde (Sigma, Catalog # P0657) 50 mg/mL (373 mM) stock in DMSO
  • 0.5 M Sodium Borate, pH 9.0
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhVNN1 to 1 µg/mL in Assay Buffer.
  2. Dilute Substrate to 500 µM in Assay Buffer.
  3. Load 50 µL of dilute rhVNN1 to empty wells of a black well plate.
  4. Start reaction by adding 50 µL of dilute substrate to wells containing enzyme. Create Enzyme Controls by not adding substrate to the wells.
  5. Seal plate with a plate sealer and incubate at 37 °C for 30 minutes.
  6. Prepare Detection mixture containing 15 mM oPA in 0.5 M sodium borate, pH 9.0.
  7. Add 100 µL Detection mixture to all wells used. For Enzyme Controls, add dilute substrate after Detection Mixture is added.
  8. Mix well and incubate sealed plate at room temperature for 5 minutes.
  9. Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively, in endpoint mode.
  10. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU)) / (Incubation time (min) x amount of enzyme (µg))

  *Adjusted for Enzyme Control
  **Derived using calibration standard Cysteamine Hydrochloride (Sigma, Catalog # M6500).

  Per Well:
  • rhVNN1: 0.050 μg
  • Substrate: 125 µM

Notes

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

Alternate Names for Recombinant Human Vanin-1/VNN1 Protein, CF

• EC 3.5.1
• EC 3.5.1.92
• HDLCQ8
• MGC116930
• MGC116931
• MGC116932
• Pantetheinase
• Pantetheine hydrolase
• Tiff66
• Tiff66MGC116933
• vanin 1
• Vanin1
• Vanin-1
• vannin 1
• Vascular non-inflammatory molecule 1
• VNN1

Background

Vanin‑1 (VNN1) is a member of the biotinidase family and is expressed at the cell surface in epithelial cells (1). VNN1 is also known as vascular non‑inflammatory molecule 1. It does not possess biotinidase activity, but is a pantetheinase that catalyzes the hydrolysis of pantetheine to pantothenic acid (vitamin‑B5) and cysteamine (2, 3). VNN1 is considered to be a potential biomarker for the acute kidney injury (4) and a target for therapeutic intervention in inflammatory bowel disease (5). Recombinant human VNN1 was engineered to have a C‑terminal truncation that prevents the normal GPI-anchor modification, resulting in its secretion.

1. Pitari, G. et al. (2000) FEBS Lett. 483:149.
2. Maras, B. et al. (1999) FEBS Lett. 461:149.
3. Martin, F. et al. (2001) Immunogenetics 53:296.
4. Hosohata, K. et al. (2012) J. Phamacol. Exp. Ther. 341:656.
5. Berruyer, C. et al. (2006) J. Exp. Med. 203:2817.

Bioinformatics

• Gene Symbol: VNN1
• Uniprot:
  • O95497()