Recombinant Human HGF Activator Protein, CF

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

Recombinant Human HGF Activator Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ (Catalog # ES002). The specific activity is >15 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human HGF Activator protein
  Gln36-Ser655, with a C-terminal 10-His tag
• Accession #: Q04756
• N-terminal Sequence: Gln36
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: HGFAC
• 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: 68 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: 105 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 CaCl₂.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Activation Buffer: 50 mM Tris, 150 mM NaCl, 10 mM CaCl₂, 10 µg/mL dextran sulfate, pH 7.5
  • Assay Buffer: 50 mM Tris, 0.01% (w/v) CHAPS, pH 9.0
  • Recombinant Human HGF Activator (rhHGFA) (Catalog # 1514-SE)
  • Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
  • 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
  • Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002), 2 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 rhHGFA to 100 µg/mL in Activation Buffer containing Thermolysin at 4.3 µg/mL.
  2. Incubate at 37 °C for 1 hour.
  3. Dilute 1,10 Phenanthroline to 20 mM in Assay Buffer.
  4. Add an equal volume of 20 mM 1,10 Phenanthroline (final concentration 10 mM) to stop Thermolysin activity.
  5. Incubate at room temperature for 30 minutes.
  6. Dilute rhHGFA to 10 ng/µL in Assay Buffer.
  7. Dilute Substrate to 20 µM in Assay Buffer.
  8. Load in a black well plate 50 µL of 10 ng/µL rhHGFA, and start the reaction by adding 50 µL of 20 µM Substrate. Prepare a Substrate Blank by combining 50 µL of 20 µM Substrate with 50 µL of Assay Buffer.
  9. Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes.
  10. 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:
  • rhHGFA: 0.5 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 HGF Activator Protein, CF

• HGF Activator
• HGFA
• Hgfac

Background

Hepatocyte Growth Factor Activator (HGFA) is a serine endopeptidase that cleaves at the peptide bond between Arg494 and Val495 of single-chain human HGF precursor, generating the active heterodimer (1). HGFA is produced and secreted by the liver and normally circulates in the blood as an inactive zymogen (2, 3). The zymogen has a weak affinity for heparin but acquires a strong affinity for heparin upon activation that is linked to blood coagulation. This property may ensure the local action of this enzyme at the site of tissue injury (3). Human HGFA precursor (655 amino acid residues) contains several predicted domains including a signal peptide (residues 1‑30), a propeptide (residues 31-372), and a mature and active form (residues 373-655) that is further processed into a short chain (residues 373‑407) and a long chain (residues 408-655). The short chain and the long chain (catalytic domain) may form a disulfide bond linked dimer. HGFA can be activated by thrombin (Catalog # 1473-SE)  or thermolysin (Catalog # 3097-ZN)  (4). The active protease can be inhibited by HGFA inhibitors (HAIs). Two HAIs, HAI-1 and HAI-2, are known in mouse and human. HAI-1 is not only an inhibitor, but also a specific acceptor of active HGFA, acting as a reservoir of this enzyme on the cell surface (5).

1. Kitamura, N. (2004) in Handbook of Proteolytic Enzymes (Barrett, A.J. et al. Eds.) p. 1712, Academic Press, San Diego.
2. Miyazawa, K. et al. (1993) J. Biol. Chem. 268:10024.
3. Miyazawa, K. et al. (1996) J. Biol. Chem. 271:3615.
4. Shimomura, T. et al. (1993) J. Biol. Chem. 268:22927.
5. Kataoka, H. et al. (2000) J. Biol. Chem. 275:40453.

Publications for HGF Activator (1514-SE)(2)

Publications using 1514-SE

• Pant S, Mukonoweshuro A, Desai B, Ramjee M, Selway C, Tarver G, Wright A, Birchall K, Chapman T, Tervonen T, Klefstrom J Design, Synthesis, and Testing of Potent, Selective Hepsin Inhibitors via Application of an Automated Closed-Loop Optimization Platform. J Med Chem, 2018-05-14;61(10):4335-4347. 2018-05-14 [PMID: 29701962] (Enzyme Assay)
• Kitajima T, Terai H, Ito Y A fusion protein of hepatocyte growth factor for immobilization to collagen. Biomaterials, 2007-01-19;28(11):1989-97. 2007-01-19 [PMID: 17239947] (Enzyme Assay, Human)

Bioinformatics

• Gene Symbol: HGFAC
• Uniprot:
  • Q04756()