Recombinant E. coli Ecotin Protein, CF

• Reactivity: Ec
• Applications: Inhibition Activity
• Format: Carrier-Free

Recombinant E. coli Ecotin Protein, CF Summary

• Details of Functionality: Measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ (Catalog # ES002). The IC₅₀ value is <1.0 nM, as measured under the described conditions.
• Source: E. coli-derived e. coli Ecotin protein
  Ala21-Arg162, with a C-terminal 6-His tag
• Accession #: NP_416713
• N-terminal Sequence: Ala21
• Protein/Peptide Type: Recombinant Enzymes
• 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:
  • Inhibition Activity
• Theoretical MW: 17 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: 18 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 reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Reconstitution Instructions: Reconstitute at 100 μg/mL in sterile 25 mM Tris and 350 mM NaCl, pH 8.5.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl₂, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Recombinant E. coli Ecotin (reEcotin) (Catalog # 1328-PI)
  • Trypsin (Sigma, Catalog # T-1426)
  • Fluorogenic Peptide 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)
  • Plate Reader (Model: SpectraMax Gemini by Molecular Devices) or equivalent
  1. Dilute Trypsin to 0.25 μg/mL in Assay Buffer.
  2. Prepare a curve of reEcotin (MW: 17,042 Da) in Assay Buffer. Make serial dilutions of: 200, 100, 40, 20, 16, 10, 4 and 1.6 nM.
  3. Combine equal volumes of the reEcotin curve dilutions and the diluted active Trypsin. Include a control (in duplicate) containing Assay Buffer and the diluted active Trypsin.
  4. Incubate mixtures at room temperature for 30 minutes.
  5. Perform 1:5 dilutions of reaction mixture with Assay Buffer.
  6. Dilute fluorogenic peptide substrate to 20 μM in Assay Buffer.
  7. Load in a black well plate 50 μL of the incubated mixtures, and start the reaction by adding 50 μL of 20 μM substrate.
  8. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.
  9. Derive the 50% inhibition concentration (IC₅₀) for reEcotin by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  10. The specific activity for Trypsin at each point may be determined using the following formula (if needed):

  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:
  • Trypsin: 0.00125 μg
  • reEcotin: 10, 5.0, 2.0, 1.0, 0.8, 0.5, 0.2, 0.08 nM
  • Substrate: 10 μM

Notes

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

Alternate Names for Recombinant E. coli Ecotin Protein, CF

• Ecotin

Background

Ecotin is a potent and general inhibitor of serine proteases with diverse substrate specificities (1). It strongly inhibits trypsin, chymotrypsin, elastase, factor Xa, plasma kallikrein, u-plasminogen activator, granzyme B, and factor XIIa. Immobilized Ecotin has been used to affinity-purify recombinant trypsinogen, indicating that it may also be used to purify additional serine protease zymogens (2). Compared to other serine protease inhibitors such as members of the serpin family, the reactive site of Ecotin is Met104 (P1) (3). Ecotin is synthesized as a 162 amino acid precursor with a 20 amino acid signal peptide necessary to direct it to the periplasmic space. The mature protein (residues 21‑162) is expressed and purified.

1. McGrath, M.E. et al. (1995) Protein Sci. 4:141.
2. Lengyel, Z. et al. (1998) Protein Expre. Purif. 12:291.
3. McGrath, M.E. et al. (1991) J. Biol. Chem. 28:6620.

Publications for Ecotin (1328-PI)(1)

Publication using 1328-PI

• Cortesio CL, Jiang W Mannan-binding lectin-associated serine protease 3 cleaves synthetic peptides and insulin-like growth factor-binding protein 5. Arch. Biochem. Biophys., 2006-03-03;449(1):164-70. 2006-03-03 [PMID: 16554018] (Enzyme Assay, Human)

Bioinformatics

• Uniprot:
  • NP_416713()