Recombinant Human Aminopeptidase P2/XPNPEP2 Protein, CF

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

Recombinant Human Aminopeptidase P2/XPNPEP2 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, H-Lys(2-Aminobenzoyl)-Pro-Pro-p-Nitroanilide (K(Abz)PP-pNA). The specific activity is >300 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Aminopeptidase P2/XPNPEP2 protein
  His22-Ala650, with a C-terminal 10-His tag
• Accession #: AAB96394
• N-terminal Sequence: His22 & Lys24
• Protein/Peptide Type: Recombinant Enzymes
• Gene: XPNPEP2
• 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: 72 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: 85 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 and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 100 mM HEPES, 0.5 mM MnCl₂, pH 8.0
  • Recombinant Human Aminopeptidase P2/XPNPEP2 (rhXPNPEP2) (Catalog # 2490-ZN)
  • Substrate: Lys(Abz)-Pro-Pro-pNA (Bachem, Catalog # L-1980), 10 mM stock in deionized water
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhXPNPEP2 to 1 ng/µL in Assay Buffer. Incubate at room temperature for 30 minutes.
  2. Dilute Substrate to 100 μM in Assay Buffer.
  3. Load into plate 50 μL of 1 ng/µL of rhXPNPEP2 and start the reaction by adding 50 µL of 100 µM Substrate. Include a Substrate Blank of 50 μL Assay Buffer and 50 µL of 100 µM Substrate.
  4. Read at excitation and emission wavelengths of 320 nm and 410 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 ABZ-Gly (Bachem, Catalog # E-2920).

  Per Well:
  • rhXPNPEP2: 0.05 µg
  • Substrate: 50 µM

Notes

The purchase of this product conveys to the buyer the limited, non-exclusive, non-transferable right (without the right to resell, repackage, or further sublicense) to use these reagents for non-commercial research purposes only. No other license is granted to the buyer whether expressly, by implication, by estoppel or otherwise. In particular, the purchase of this product does not include nor carry any right or license to use, develop, or otherwise exploit this product commercially, which includes without limitation, provision of services to a third party, generation of commercial databases, clinical diagnostics or therapeutics, or drug development. This Product is manufactured under a license to U.S. Patent Nos. 6,399,349 and 7,273,718.

Any party desiring rights under this patent should contact Ryogen LLC, Montebello Park, 75 Montebello Road, Suffern, NY 10901.

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

Alternate Names for Recombinant Human Aminopeptidase P2/XPNPEP2 Protein, CF

• Aminoacylproline aminopeptidase
• Aminopeptidase P2
• APP2
• EC 3.4.11.9
• mAmP
• Membrane-bound aminopeptidase P
• Membrane-bound AmP
• Membrane-bound APP
• xaa-Pro aminopeptidase 2
• XPNPEP2
• X-Pro aminopeptidase 2
• X-prolyl aminopeptidase (aminopeptidase P) 2, membrane-bound

Background

The human XPNPEP2 gene encodes Aminopeptidase P2 (APP2), which is also known as X-prolyl Aminopeptidase 2 or membrane bound Aminopeptidase P (1‑4). It is a member of the M24 family of metalloproteases, which also contains methionine Aminopeptidases, X-Pro dipeptidase, Aminopeptidase P1, Aminopeptidase P homolog, proliferation-associated protein 1, and suppressor of Ty homolog or chromatin-specific transcription elongation factor large subunit (5). Mammalian APP2 are predicted to be GPI-anchored membrane proteases and their biological functions have been reviewed (6). Human APP2 is widely expressed in many adult tissues with the highest levels in the kidney (7). The purified recombinant human APP2 corresponds to the ectodomain and is shown here to be an active aminopeptidase, removing a N-terminal amino acid from a peptide that contains a Pro residue at the second position.

1. Venema, R.C. et al. (1997) Biochim. Biophys. Acta 1354:45.
2. Sprinkle, T.J. et al. (1998) Genomics 50:114.
3. Cottrell, G.S. et al. (1998) Biochem. Soc. Trans. 26:S248.
4. Prueitt, R.L. et al. (2000) Cytogenet. Cell Genet. 89:44.
5. Barrett, A.J. et al. (2004) Handbook of Proteolytic Enzymes, Elsevier Academic Press, San Diego.
6. Simmons, W.H. (2004) in Handbook of Proteolytic Enzymes (Barrett, A.J. et al. eds.) p. 934, Elsevier Academic Press, San Diego.
7. Ersahin C. et al. (2005) Arch. Biochem. Biophys. 435:303.

Bioinformatics

• Gene Symbol: XPNPEP2
• Uniprot:
  • AAB96394()