Recombinant Human Neprilysin-2/MMEL1 Protein, CF

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

Recombinant Human Neprilysin-2/MMEL1 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 >250 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Neprilysin-2/MMEL1 protein
  Gly69-Trp770, with an N-terminal 6-His tag
• Accession #: AAL08942
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: MMEL1
• 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: 82 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: 99 kDa, 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 opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and ZnCl₂.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 250 mM NaCl, pH 7.5
  • Recombinant Human Neprilysin-2/MMEL1 (rhNeprilysin-2) (Catalog # 2340-ZN)
  • Substrate: MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH (Catalog # ES005)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhNeprilysin-2 to 1 µg/mL in Assay Buffer.
  2. Dilute Substrate to 20 µM in Assay Buffer.
  3. Load into a black well plate 50 µL of 1 µg/mL rhNeprilysin-2, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 20 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:
  • rhNeprilysin-2: 0.050 µg
  • Substrate: 10 µM

Notes

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

Alternate Names for Recombinant Human Neprilysin-2/MMEL1 Protein, CF

• EC 3.4.24
• EC 3.4.24.11
• MELL1
• membrane metallo-endopeptidase-like 1
• Membrane metallo-endopeptidase-like 2MGC119455
• MGC119454
• MMEL1
• MMEL2
• NEP2
• NEP2(m)
• NEPIIMGC119456
• Neprilysin II
• Neprilysin2
• Neprilysin-2
• NL2
• NL2NL1
• SEP
• soluble secreted endopeptidase
• zinc metallopeptidase

Background

Neprilysin-2 is a zinc protease of the neprilysin (NEP) family, which also includes NEP, ECE-2, PEX, DINE, Kell and several NEP-like proteins (1). It is encoded by the MMEL1 gene in the human genome and is also known as soluble secreted endopeptidase (SEP), Neprilysin-like 1 and Neprilysin-2 (2). Highly expressed in testis, the cDNA predicted a type II transmembrane protein with a short cytoplasmic tail and a large ectodomain. Both membrane-bound and soluble forms were comparable with regard to model substrates, pH optima and inhibitor profiles. The ectodomain of human Neprilysin-2/MMEL1 was expressed with an N-terminal His tag and purified.

1. Turner, A.J. et al. (2001) BioEssays 23:261.
2. Carpentier, M. et al. (2004) in Handbook of Proteolytic Enzymes. Barrett, A.J. et al. eds. p. 426.

Publications for Neprilysin-2/MMEL1 (2340-ZN)(1)

Publication using 2340-ZN

• Huang JY, Bruno AM, Patel CA, Huynh AM, Philibert KD, Glucksman MJ, Marr RA Human membrane metallo-endopeptidase-like protein degrades both beta-amyloid 42 and beta-amyloid 40. Neuroscience, 2008-05-16;155(1):258-62. 2008-05-16 [PMID: 18571334] (Enzyme Assay, Western Blot, Human)

Bioinformatics

• Gene Symbol: MMEL1
• Uniprot:
  • AAL08942()