Recombinant Cynomolgus Aminopeptidase PILS/ARTS1 Protein, CF

Recombinant Cynomolgus Monkey Aminopeptidase PILS/ARTS1 His-tag (Catalog # 10237-ZN) is measured by its ability to cleave the fluorogenic peptide substrate, Leu-AMC.

2 μg/lane of Recombinant Cynomolgus Monkey Aminopeptidase PILS/ARTS1 His-tag was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing a band under reducing conditions at 101 kDa.

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

Recombinant Cynomolgus Aminopeptidase PILS/ARTS1 Protein, CF Summary

• Additional Information: His-tag
• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, Leu-AMC. The specific activity is >800 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived cynomolgus monkey Aminopeptidase PILS/ARTS1 protein
  Ala37-Leu941
  with a C-terminal 6-His tag
• Accession #: XP_005557484
• N-terminal Sequence: Ala37
• 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: 104 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: 95-107 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 °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 visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 25 mM Tris, pH 8.0
  • Recombinant Cynomolgus Monkey Aminopeptidase PILS/ARTS1 His-tag (cynoARTS1) (Catalog # 10237-ZN)
  • Substrate: Leu-AMC (Bachem, Catalog # I-1240), 10 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rcynoARTS1 to 1 µg/mL in Assay Buffer.
  2. Dilute Substrate to 600 µM in Assay Buffer.
  3. Load in plate 50 µL of 1 µg/mL rcynoARTS1, and start the reaction by adding 50 µL of 600 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 600 µM Substrate.
  4. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode of 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 7-amino, 4-Methyl Coumarin AMC (Sigma, Catalog # A9891)

  Per Well:
  • rcynoARTS1: 0.05 µg
  • Substrate: 300 µM

Notes

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

Alternate Names for Recombinant Cynomolgus Aminopeptidase PILS/ARTS1 Protein, CF

• Adipocyte-derived leucine aminopeptidase
• A-LAP
• A-LAPALAP
• Aminopeptidase PILS
• ARTS1
• ARTS1aminopeptidase regulator of TNFR1 shedding
• ARTS-1PILSAP
• EC 3.4.11
• EC 3.4.11.-
• EC 3.4.11.1
• endoplasmic reticulum aminopeptidase 1
• endoplasmic reticulum aminopeptidase associated with antigen processing
• ERAAP1
• ERAP1
• KIAA0525APPILS
• PILS-AP
• PILS-APERAAP
• Puromycin-insensitive leucyl-specific aminopeptidase
• Type 1 tumor necrosis factor receptor shedding aminopeptidase regulator

Background

ARTS1 encodes a zinc-dependent metalloprotease (1) known as aminopeptidase PILS (Puromycin-Insensitive Leucyl-Specific), ALAP (adipocyte-derived leucine aminopeptidase), TNFR (type 1 tumor necrosis factor receptor) shedding aminopeptidase regulator, or ERAP1 (endoplasmic reticulum aminopeptidase 1). ARTS1 is widely expressed and known to be present in the endoplasmic reticulum. The protein has four domains with a large cavity between domain II and IV that can accommodate large peptide substrates; the second domain contains the active site and the first domain caps the active site and provides binding sites for the amino terminus of the substrate peptides (2). The enzyme has a unique substrate preference amongst the M1-family of aminopeptidases for longer peptides of up to 16 residues (3, 4) that is consistent with its ability to trim antigen precursors for MHC class 1 presentation (5). Variants and polymorphisms identified in ARTS-1 have linked it to autoimmune disease (6), cancer (7), and hypertension (8). ARTS-1 has been shown to be secreted (9, 10) and cause activation of inflammasome and cathepsin B pathways (11) and consequently to have a direct role in innate immunity and contribute to autoinflammatory and autoimmune diseases (12). ARTS-1 is consequently a potential therapeutic target (12-14).

1. Schomburg, L. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, et al.) pp. 311, Academic Press, San Diego.
2. Nguyen, T. T. et al. (2011) Nat. Struct. Mol. Biol. 18:604.
3. York, I. A. et al. (2002) Nat. Immunol. 3:1177.
4. Chang, S. C. et al. (2005) Proc. Nat. Acad. Sci. U.S.A. 102:17107.
5. Serwold, T. et al. (2002) Nature 419:480.
6. Fung, E. Y. et al. (2009) Genes Immun. 10:188.
7. Mehta, A. M. et al. (2009) Genes Chromosomes Cancer 48:410.
8. Yamamoto, N. et al. (2002) Hum. Mutat. 19:251.
9. Cui, X. et al. (2002) J. Clin. Invest. 110:515.
10. Goto, Y. et al. (2011) J. Biol. Chem. 286:21906.
11. Aldhamen, Y. A. et al. (2015) J. Innate Immun. 7:275.
12. Pepelyayeva, Y. and A. Amalfitano. (2019) Hum. Immunol. 80:302.
13. Koumantou, D. et al. (2019) Cancer Immunol. Immunother. 68:1245.
    
14. Georgiadis, D. et al. (2018) Curr. Med. Chem. [Epub ahead of print]
    

Bioinformatics

• Uniprot:
  • XP_005557484()