Recombinant Cyno u-Plasminogen Activator (uPA) Protein, CF

Recombinant Cynomolgous Monkey u-Plasminogen Activator (uPA)/Urokinase His-tag (Catalog # 11098-SE) is measured by its ability to cleave a peptide substrate, N-carbobenzyloxy-Gly-Gly-Arg-7-amido-4-methylcoumarin (Z-GGR-AMC).

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

Recombinant Cyno u-Plasminogen Activator (uPA) Protein, CF Summary

• Additional Information: His-tag
• Details of Functionality: Measured by its ability to cleave a peptide substrate, N-carbobenzyloxy-Gly-Gly-Arg-7-amido-4-methylcoumarin (Z-GGR-AMC). The specific activity is >2000 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived cynomolgus monkey u-Plasminogen Activator (uPA)/Urokinase protein
  Ser21-Leu430 with a C-terminal 10-His tag
• Accession #: XP_05565501.1
• N-terminal Sequence: Lys155, Ile178, Met358
• 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: 18 kDa (long A chain), 3 kDa (short A chain), 30 kDa (B chain).
  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: 32-41 kDa (short form) and 48-59 kDa (long form), non-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 HEPES, NaCl and CaCl₂.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 0.01% (v/v) Tween® 20, pH 8.5
  • Recombinant Cynomolgus Monkey u-Plasminogen Activator (uPA)/Urokinase (rcyno uPA) (Catalog # 11098-SE)
  • Substrate: Z-Gly-Gly-Arg-AMC (Bachem, Catalog # I-1140), 10 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax M5 by Molecular Devices) or equivalent
  1. Dilute rcyno uPA to 1 ng/µL in Assay Buffer.
  2. Dilute Substrate to 400 µM in Assay Buffer.
  3. Load 50 μL of 1 ng/µL rcyno uPA into a black well plate, and start the reaction by adding 50 µL of 400 µM Substrate. Include a Substrate Blank containing 50 µL of Assay buffer and 50 µL of 400 µM Substrate.
  4. Read at excitation and emission wavelengths of 380 nm and 460 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 from calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A9891)
  Per Reaction:
  • rcyno uPA: 0.05 µg
  • Substrate: 200 µM

Notes

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

Alternate Names for Recombinant Cyno u-Plasminogen Activator (uPA) Protein, CF

• ATF
• EC 3.4.21
• EC 3.4.21.73
• plasminogen activator, urokinase
• PLAU
• uPA
• u-PA
• uPlasminogen Activator
• u-Plasminogen Activator
• urinary
• Urokinase
• urokinase-type plasminogen activator

Background

Urokinase Plasminogen Activator (uPA), also known as u-plasminogen activator or urokinase, is a highly-specific serine protease from the peptidase S1 family that cleaves plasminogen to form plasmin making it a key player in the plasminogen activator (PA) system (1, 2). In cancer, the PA system plays a commanding role in tumor growth, angiogenesis, tumor cell invasion, migration, and metastasis. Expression of uPA is minimal in normal cells but is increased several fold in tumor cells by extracellular stimuli elevated in cancer (3) and corresponds to poor outcomes in several types of cancer (2, 4-7). Therefore, uPA has been identified as an excellent target for therapeutic development through inhibition of protease activity or though inhibition of uPA-dependent signaling while in complex with uPA receptor (uPAR) (2, 7). The pro-enzyme of uPA is synthesized with an N-terminal signal peptide and processed into an active disulfide-linked two-chain molecule (2, 7-10). For the cynomolgous uPA, the B chain starting at Ile178 corresponds to the catalytic domain. Two forms of the A chain exist, one starting at Ser21 (the long form) and the other at Lys155 (the short form). While the B chain is common for both forms, the long and short A chains are unique to expected 48 kDa and 32 kDa two-chain forms, respectively. The long A chain contains an EGF-like domain and the kringle domain. The long A domain is reportedly responsible for the binding of the uPA receptor (uPAR) (2,7). Both high and low MW forms exist in the purified recombinant cyno uPA.

1. Ellis, V. (2004) in Handbook of Proteolytic Enzymes. Barrett, A.J. et al. eds. Academic Press, San Diego, pp.1677. 
2. Mahmood, N. et al. (2018) Front Oncol. 8:24.
3. Nagamine, Y. et al. (2005) Thromb. Haemost. 93:661.
4. Duffy, M. and C. Duggan (2004) Clin. Biochem. 37:541.
5. Pappot, H. et al. (2006) Lung Cancer 51:193.
6. Taubert, H. et al. (2010) Br. J. Cancer 102:731.
7. Masucci, M.T. et al. (2022) Cancers. 14:498.
8. Riccio, A. et al. (1985) Nucleic Acids Res. 13:2759.
9. Nagai, M. et al. (1985) Gene 36:183.
10. Jacobs, P. et al. (1985) DNA 4:139.

Bioinformatics

• Uniprot:
  • XP_05565501.1()