Recombinant Cynomolgus/Rhesus Neprilysin/CD10 His Protein CF

Recombinant Cynomologous Monkey/Rhesus Macaque Neprilysin His-tag Protein (Catalog # 10834-ZN) is measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPPGFSAFK(Dnp)-OH (ES005).

2 μg/lane of Recombinant Cynomolgus Monkey/Rhesus Macaque Neprilysin/CD10 His-tag Protein (Catalog # 10834-ZN) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 86-95 kDa.

• Reactivity: Pm-Cm, RM
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Cynomolgus/Rhesus Neprilysin/CD10 His Protein CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPPGFSAFK(Dnp)-OH (Catalog # ES005). The specific activity is >1200 pmol/min/μg, as measured under the described conditions.
• Source: Human embryonic kidney cell, HEK293-derived Neprilysin/CD10 protein Tyr52-Trp750 with an N-terminal 6-His tag
• Accession #: XP_005546216.1
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 81 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: 86-95 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 degreesC as supplied. 3 months, -20 to -70 degreesC under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and ZnCl 2 .
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 0.5 M NaCl, pH 9.0
  • Recombinant Cynomolgus/Rhesus Neprilysin (rcyno/rhesusNeprilysin) (Catalog # 10834-ZN)
  • Substrate: Mca-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH (Catalog # ES005), 2 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rcyno/rhesusNeprilysin to 0.2 µg/mL in Assay Buffer.
  2. Dilute Substrate to 60 µM in Assay Buffer.
  3. Load into a plate 50 µL of 0.2 µg/mL rcyno/rhesusNeprilysin and start the reaction by adding 50 µL of 60 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 60 µM Substrate.
  4. Read at excitation and emission wavelengths of 320 nm and 405 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 MCA-Pro-Leu-OH (Bachem, Catalog # M-1975)

  Per Well:
  • rcyno/rhesusNeprilysin: 0.01 µg
  • Substrate: 30 µM

Notes

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

Alternate Names for Recombinant Cynomolgus/Rhesus Neprilysin/CD10 His Protein CF

• Atriopeptidase
• CALLA
• CALLAmembrane metallo-endopeptidase (neutral endopeptidase, enkephalinase)
• CD10 antigen
• CD10
• CD10)
• CD10membrane metallo-endopeptidase variant 1
• Common acute lymphocytic leukemia antigen
• DKFZp686O16152
• EC 3.4.24
• EC 3.4.24.11
• Enkephalinase
• EPN
• Leu-19
• membrane metallo-endopeptidase variant 2
• membrane metallo-endopeptidase
• MGC126681
• MGC126707
• MME
• NEPmembrane metallo-endopeptidase (neutral endopeptidase, enkephalinase, CALLA
• Neprilysin
• Neutral Endopeptidase 24.11
• Neutral endopeptidase
• NKH1
• SFE
• Skin fibroblast elastase

Background

Neprilysin/CD10, also known as NEP, neutral endopeptidase, enkephalinase, and common acute lymphocytic leukemia antigen (CALLA), is a zinc-dependent metallopeptidase type II integral membrane protein of the M13 subfamily of neutral endopeptidases. The enzyme functions both as an endopeptidase with a thermolysin-like specificity and as a dipeptidylcarboxypeptidase. It cleaves and degrades a variety of bioactive peptides including natriuretic peptides, enkephalins, and tachykinins at the amino side of hydrophobic residues. Neprilysin is widely distributed in tissues and highly conserved among mammals (1). Neprilysin is a noncovalently associated homodimer where the monomer is composed of a short intracellular domain, transmembrane helix, and large C-terminal extracellular domain including two domains that form a central cavity containing the active site (2). Although a membrane protein, an alternatively processed soluble form has been documented in circulation and retains similar enzymatic activity to the membrane bound Neprilysin (3). The soluble form is of interest as a prognostic biomarker for heart failure (4,5) and inhibition of Neprilysin is a therapeutic strategy for heart failure (6). Neprilysin was found to be a major degrading enzyme of amyloid beta peptide (A beta) in the brain, making it an important target in Alzheimer's disease studies (7). It metabolizes sensory and inflammatory neuropeptides making it a target for pain perception (8) and is an important marker for some types of lymphomas (9,10). There is also interest in Neprilysin as a biomarker (11) and as a therapeutic inhibition target for treatment of type 2 diabetes (12).

1. Chen, Y. et al. (2017) Clin. Chem. 63:108.
2. Oefner, C. et al. (2000) J. Mol. Biol. 296:341.
3. Aviv, R. et al. (1995) Kidney Int. 47:855.
4. Bayes-Genis, A. et al. (2015) J. Am. Coll. Cardiol. 65:657. 
5. Goliasch, G. et al. (2016) Eur. J. Heart Fail. 18:89. 
6. Von Lueder, T.G. et al. (2013) Circ. Heart Fail. 6:594.
7. Iwata, N. et al. (2001) Science 292:1550.
8. Turner, A.J. et al. (2007) Int. Rev. Neurobiol. 82:113. 
9. De Jong, D. et al. (2007) J. Clin. Oncol. 25:805.
10. Yang, X.L. et al. (2017) Chin. Med. J. 130:1973.
11. Gutta, S. et al. (2018) Am. J. Physiol. Renal. Physiol. 315:F263.
12. Esser, N. et al. (2019) Diabetologia 62:1113.

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Array 10834-ZN

• Neprilysin/CD10 Antibodies
• Neprilysin/CD10 ELISA Kit
• Neprilysin/CD10 Lysates
• Neprilysin/CD10 Proteins
• Neprilysin/CD10 Proteins and Enzymes
• Neprilysin/CD10 ELISAs

Bioinformatics

• Uniprot:
  • XP_005546216.1()