Recombinant Cynomolgus 5'-Nucleotidase/CD73 His Protein, CF

R&D Systems | Catalog # 10173-EN

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Key Product Details

  • R&D Systems CHO-derived Recombinant Cynomolgus 5'-Nucleotidase/CD73 His Protein (10173-EN)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

CHO

Accession Number

XP_005552488.1

Structure / Form

Dimer

Applications

Enzyme Activity
View all 5'-Nucleotidase/CD73 Proteins and Enzymes »

Product Specifications

Source

Chinese Hamster Ovary cell line, CHO-derived cynomolgus monkey 5'-Nucleotidase/CD73 protein
Trp27-Lys547
with a C-terminal 6-His tag

Purity

>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

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

N-terminal Sequence Analysis

Trp27

Predicted Molecular Mass

59 kDa

SDS-PAGE

57-75 kDa

Activity

Measured by its ability to hydrolyze the 5’-phosphate group from the substrate adenosine-5’-monophosphate (AMP). The orthophosphate product is measured by a Malachite Green Phosphate Detection Kit (Catalog # DY996).
The specific activity is >6500 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

10173-EN
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, CaCl2 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.

Background: 5'-Nucleotidase/CD73

CD73, known as ecto-5'-Nucleotidase, converts extracellular nucleoside 5' monophosphates to nucleosides, with AMP as its preferred substrate (1). CD73 is a zinc-dependent, 70 kDa homodimeric enzyme bound to the cell membrane through a glycosyl phophatidylinositol (GPI) anchor. It is composed of an N-terminal domain containing metal binding sites linked via small hinge region to a C-terminal domain containing the substrate binding site and dimerization interface (2). It is expressed by most cell types (3) and is widely expressed in tumor cell lines as well as upregulated in cancerous tissues (4,5). CD73 is a key enzyme responsible for a rate-limiting step in the generation of extracellular adenosine. Adenosine is a molecule that signals through activation of purinergic receptors and results in an immunosuppressive role in the tumor microenvironment (4,6). CD73 has been implicated in many pathological processes including immunomodulation and inflammation (7,8) tumor growth and metastasis (9-13) making CD73 a potential drug target in cancer. Targeting CD73 inhibition has resulted in numerous reports of favorable antitumor effects (4,5,12). Consequently, therapeutic approaches have been tested using knockdown, gene silencing and anti-CD73 therapies (11,14) as well as small molecule inhibitors (14,15).

References

  1. Zimmermann, H. et al. (2012) Purinergic Signal. 8:437.
  2. Knapp, K. et al. (2012) Structure. 20:2161.
  3. Resta, R. et al. (1998) Immunol. Rev. 161:95.
  4. Jin, D. et al. (2010) Cancer Res. 70:2245.
  5. Zhang, B. (2010) Cancer Res. 70:6407.
  6. Pilcher, M. et al. (2003) J. Biol. Chem. 278:13468.
  7. Antonioli, L. et al. (2012) Curr. Drug Targets 13:842.
  8. Eltzchig, H. K. et al. (2012) N. Engl. J. Med. 367:2322.
  9. Bavaresco, L. et al. (2008) Mol. Cell Biochem. 319:61.
  10. Yegutkin, G. G. et al. (2011) Eur. J. Immunol. 41:1231.
  11. Stagg, J. et al. (2012) Cancer Res. 72:2190.
  12. Ghalamfarsa, G. et al. (2019) Expert Opin. Ther. Targets. 23:127.
  13. Gao, Z. W. et al. (2014) Biomed. Res. Int. [PMID 25126561].
  14. Young, A. et al. (2014) Cancer Discov. 4:879.
  15. McManus, J. et al. (2018) SLAS Discov. 23:264.

Alternate Names

5-NT, CD73, E5NT, eNT, NT5E, NTE

Entrez Gene IDs

4907 (Human); 23959 (Mouse); 58813 (Rat); 102114976 (Cynomolgus Monkey)

Gene Symbol

NT5E

UniProt

XP_005552488.1

Additional 5'-Nucleotidase/CD73 Products

Product Documents for Recombinant Cynomolgus 5'-Nucleotidase/CD73 His Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Cynomolgus 5'-Nucleotidase/CD73 His Protein, CF

For research use only

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Protocols

View specific protocols for Recombinant Cynomolgus 5'-Nucleotidase/CD73 His Protein, CF (10173-EN):

Materials
  • Assay Buffer: 25 mM Tris, 5 mM MgCl2, pH 7.5
  • Recombinant Cynomolgus Monkey 5'‑Nucleotidase/CD73 (rcynoCD73) (Catalog # 10173-EN)
  • Adenosine monophosphate (AMP) (Sigma, Catalog # A1752), 5 mM stock in deionized water
  • Malachite Green Phosphate Detection Kit (Catalog # DY996)
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare a standard curve from the 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of Assay Buffer for a 10 mM stock. Continue by adding 10 µL of the 10 mM Phosphate stock to 990 µL of Assay Buffer for a 100 µM stock (this is the first dilution to use as a standard).
  2. Perform six additional one-half serial dilutions of the 100 µM Phosphate stock using Assay Buffer.  The standard curve has a range of 0.078 to 5 nmoles per well.
  3. Dilute Substrate to 100 µM in Assay Buffer.
  4. Dilute rcynoCD73 to 0.08 µg/mL in Assay Buffer.
  5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  6. Load 25 µL of 0.08 µg/mL rcynoCD73 into empty wells of the same plate as the curve. Include a Control containing 25 μL of Assay Buffer.
  7. Add 25 µL of diluted Substrate to all wells, excluding the standard curve. 
  8. Seal plate and incubate at 37 °C for 20 minutes.
  9. Add 30 µL of the Malachite Green Reagent A to all wells used, including standard curve.  Mix briefly.
  10. Add 100 µL of deionized water to all wells used, including standard curve.  Mix briefly.
  11. Add 30 µL of the Malachite Green Reagent B to all wells used, including standard curve.  Mix briefly.
  12. Seal plate and incubate at room temperature for 20 minutes.
  13. Read plate at 620 nm (absorbance) in endpoint mode.
  14. Calculate specific activity:

     Specific Activity (pmol/min/µg) =

Phosphate released* (nmol) x (1000 pmol/nmol)
Incubation time (min) x amount of enzyme (µg)

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.

Per Reaction:

  • rcynoCD73: 0.002 µg
  • Substrate: 50 µM

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