Recombinant Mouse Kallikrein 7 Protein, CF

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

Recombinant Mouse Kallikrein 7 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 >70 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse Kallikrein 7 protein
  Gln22-Arg249 with a C-terminal 6-His tag
• Accession #: Q91VE3
• N-terminal Sequence: No results obtained: Gln22 predicted
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Klk7
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 26 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: 27-37 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 HEPES and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Activation Buffer: 50 mM Tris, 150 mM NaCl, 10 mM CaCl₂, 0.05% Brij-35, pH 7.5 (TCNB)
  • Assay Buffer: 50 mM Tris, 1 M NaCl, pH 8.0
  • Recombinant Mouse Kallikrein 7 (rmKLK7) (Catalog # 7688-SE)
  • Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
  • EDTA, Disodium salt (Sigma, Catalog # E-4884) 500 mM stock in deionized water
  • Substrate: Mca-RPKPVE-NVal-WRK(Dnp)-NH₂ (Catalog # ES002)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rmKLK7 to 200 µg/mL in Activation Buffer.
  2. Dilute Thermolysin to 20 µg/mL in Activation Buffer.
  3. Activate rmKLK7 by combining equal volumes of 200 µg/mL rmKLK7 with 20 µg/mL Thermolysin.
  4. Incubate reactions at 37 °C for two hours.
  5. Stop reactions by adding EDTA to a final concentration of 50 mM.
  6. Dilute activated rmKLK7 to 2 µg/mL in Assay Buffer.
  7. Dilute Substrate to 20 µM in Assay Buffer.
  8. In a plate, load 50 µL of activated rmKLK7. Include an enzyme blank containing 50 µL Assay Buffer.
  9. Start reaction by adding 50 µL of 20 µM Substrate.
  10. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  11. 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:
  • rmKLK7: 0.1 µg
  • Substrate: 10 µM

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

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

Alternate Names for Recombinant Mouse Kallikrein 7 Protein, CF

• EC 3.4.21
• EC 3.4.21.117
• hK7
• hSCCE
• kallikrein 7 (chymotryptic, stratum corneum)
• Kallikrein 7
• kallikrein-related peptidase 7
• KLK7
• protease, serine, 6
• PRSS6
• SCCEkallikrein-7
• Serine protease 6
• signal protein
• Stratum corneum chymotryptic enzyme

Background

Kallikrein 7 (KLK7), also known as stratum corneum chymotryptic enzyme (SCCE), is a member of the tissue kallikrein family.  Predominantly expressed in the skin, a major physiological function of KLK7 is to regulate the desquamation process through proteolysis of the intercellular adhesive structures between corneocytes (1). Thus, it is involved in some inflammatory skin diseases, such as psoriasis and chronic itchy dermatitis (2, 3). Studies have shown that one potential physiological activator for KLK7 is KLK5, another member of the tissue kallikrein family. Along with KLK14, these three kallikreins form a proteolytic cascade in the stratum corneum (4). Mouse KLK7 is synthesized as a 249 amino acid precursor with a 21 amino acid signal peptide. The secreted protein has a short propeptide (residues 22‑25) and a mature chain comprised of residues 26‑249. The purified, secreted rmKLK7 corresponds to the pro form. After activation by thermolysin, it displays enzymatic activity against a fluorogenic synthetic peptide as described in the Activity Assay Protocol.

1. Caubet, C. et al. (2004) J. Invest. Dermatol. 122:1235.
2. Ekholm, E. and Egelrud, T. (1999) Arch. Dermatol. Res. 291:195.
3. Hansson, L. et al. (2002) J. Invest. Dermatol. 118:444.
4. Brattsand, M. et al. (2004) J. Invest. Dermatol. 124:198.

Bioinformatics

• Gene Symbol: Klk7
• Uniprot:
  • Q91VE3(Mouse)