Recombinant Human HGF Activator Protein, CF Summary
Product Specifications
Gln36-Ser655, with a C-terminal 10-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
1514-SE
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and CaCl2. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
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
- Dilute rhHGFA to 100 µg/mL in Activation Buffer containing Thermolysin at 4.3 µg/mL.
- Incubate at 37 °C for 1 hour.
- Dilute 1,10 Phenanthroline to 20 mM in Assay Buffer.
- Add an equal volume of 20 mM 1,10 Phenanthroline (final concentration 10 mM) to stop Thermolysin activity.
- Incubate at room temperature for 30 minutes.
- Dilute rhHGFA to 10 ng/µL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- 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.
- Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes.
- 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).
- rhHGFA: 0.5 µg
- Substrate: 10 µM
Reconstitution Calculator
Background: HGF Activator
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).
- Kitamura, N. (2004) in Handbook of Proteolytic Enzymes (Barrett, A.J. et al. Eds.) p. 1712, Academic Press, San Diego.
- Miyazawa, K. et al. (1993) J. Biol. Chem. 268:10024.
- Miyazawa, K. et al. (1996) J. Biol. Chem. 271:3615.
- Shimomura, T. et al. (1993) J. Biol. Chem. 268:22927.
- Kataoka, H. et al. (2000) J. Biol. Chem. 275:40453.
Citations for Recombinant Human HGF Activator Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 2
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Design, Synthesis, and Testing of Potent, Selective Hepsin Inhibitors via Application of an Automated Closed-Loop Optimization Platform.
Authors: Pant S, Mukonoweshuro A, Desai B, Ramjee M, Selway C, Tarver G, Wright A, Birchall K, Chapman T, Tervonen T, Klefstrom J
J Med Chem, 2018-05-14;61(10):4335-4347.
Applications: Enzyme Assay -
A fusion protein of hepatocyte growth factor for immobilization to collagen.
Authors: Kitajima T, Terai H, Ito Y
Biomaterials, 2007-01-19;28(11):1989-97.
Species: Human
Sample Types: Recombinant Protein
Applications: Enzyme Assay
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