Recombinant Human Aminopeptidase P1/XPNPEP1 Protein, CF

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

Recombinant Human Aminopeptidase P1/XPNPEP1 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 >1,300 pmol/min/µg, as measured under the described conditions.
• Source: E. coli-derived human Aminopeptidase P1/XPNPEP1 protein
  Met1-His623 & Pro2-His623, both with a C-terminal 6-His tag
• Accession #: NP_065116
• N-terminal Sequence: Met1 & Pro2
• Protein/Peptide Type: Recombinant Enzymes
• Gene: XPNPEP1
• 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: 71 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: 71 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 Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 250 mM NaCl, 0.5 mM MnCl₂, pH 8.0
  • Recombinant Human Aminopeptidase P1/XPNPEP1 (rhXPNPEP1) (Catalog # 2970-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 rhXPNPEP1 to 0.5 ng/µL in Assay Buffer.
  2. Dilute Substrate to 100 µM in Assay Buffer.
  3. Load into plate 50 µL of 0.5 ng/µL of rhXPNPEP1 (25 ng/well), 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:
  • rhXPNPEP1: 0.025 µg
  • Substrate: 50 µM

Notes

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

Alternate Names for Recombinant Human Aminopeptidase P1/XPNPEP1 Protein, CF

• Aminoacylproline aminopeptidase
• Aminopeptidase P1
• APP1
• Cytosolic aminopeptidase P
• cytosolic
• EC 3.4.11.9
• SAMP
• xaa-Pro aminopeptidase 1
• XPNPEP1
• XPNPEPLX-prolyl aminopeptidase (aminopeptidase P)-like
• X-prolyl aminopeptidase (aminopeptidase P) 1, soluble
• X-prolyl aminopeptidase 1, soluble

Background

The human XPNPEP1 gene encodes aminopeptidase P1 (APP1), which is also known as X-prolyl aminopeptidase with gene aliases of SAMP, XPNPEP, XPNPEPL, and XPNPEPL1 (1‑3). It is a member of the M24 family of metalloproteases, which also contains methionine aminopeptidases, X-Pro dipeptidase, aminopeptidase P2, aminopeptidase P homolog, proliferation-associated protein 1, and suppressor of Ty homolog or chromatin-specific transcription elongation factor large subunit (4). It is a soluble enzyme, in contrast to the GPI-anchored APP2 encoded by XPNPEP2 (5). Human APP1 is widely expressed (3). The purified rhAPP1 is an active aminopeptidase, removing a N‑terminal amino acid from a peptide that contains a Pro residue at the second position. The amino acid sequence of human APP1 is 99%, 97%, 95%, 74% and 73% identical to that of canine, bovine, mouse/rat, Xenopus and zebrafish, respectively.

1. Cottrell, G.S. et al. (2000) Biochemistry 39:15121.
2. Sprinkle, T.J. et al. (2000) Arch. Biochem. Biophys. 378:51.
3. Vanhoof, G. et al. (1997) Cytogenet. Cell Genet. 78:275.
4. Turner, A.J. and G.S. Cottrell (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, A.J. et al.) pp. 931 - 933, Elsevier Academic Press, San Diego. 
5. Simmons, W.H. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, A.J. et al.) pp. 934 - 937, Elsevier Academic Press, San Diego. 

Bioinformatics

• Gene Symbol: XPNPEP1
• Uniprot:
  • NP_065116()