Recombinant Human Kallikrein 7 Protein, CF

Recombinant Human Kallikrein 7 (Catalog # 2624‑SE) is measured byits ability to cleave the fluorogenic peptide substrate Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (Catalog # ES002).

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

Recombinant Human 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 >150 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Kallikrein 7 protein
  Glu23-His252, with a C-terminal 10-His tag
• Accession #: P49862
• N-terminal Sequence: Glu23
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: KLK7
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• 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: 29 kDa and 32 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 visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Assay Buffer: 50 mM Tris, 150 mM NaCl, pH 8.5
  • Recombinant Human Kallikrein 7 (rhKLK7) (Catalog # 2624-SE)
  • Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
  • EDTA (Sigma, Catalog # E-4884)
  • Substrate: MCA-Arg-Pro Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys(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 rhKLK7 to 200 μg/mL in Activation Buffer.
  2. Dilute Thermolysin to 20 μg/mL in Activation Buffer.
  3. Combine equal volumes of 200 μg/mL rhKLK7 and 20 μg/mL Thermolysin.
  4. Incubate at 37 °C for 2 hours.
  5. Stop reaction with an equal volume 100 mM EDTA diluted in Assay Buffer (C_f = 50 mM).
  6. Dilute activated rhKLK7 to 4 ng/μL in Assay Buffer.
  7. Dilute Substrate to 20 μM in Assay Buffer.
  8. Load 50 μL of 4 ng/μL rhKLK7 in a plate, and start the reaction by adding 50 μL of 20 μM Substrate to each well. Include a Substrate Blank containing 50 μL Assay Buffer and 50 μL of 20 μM Substrate.
  9. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), 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:
  • rhKLK7: 0.2 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 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

Human tissue Kallikrein 7 (hK7), also known as stratum corneum chymotryptic enzyme (SCCE), is a member of the human tissue kallikrein family. Full-length hK7 consists of 253 amino acids, with a signal peptide (residues 1‑22), short pro peptide (residues 23‑29) and mature chain (residues 30‑252) (1). Predominantly expressed in the skin, a major physiological function of hK7 is to regulate the desquamation process through proteolysis of the intercellular adhesive structures between corneocytes (2). Thus, it is related to some inflammatory skin diseases, such as psoriasis and chronic itchy dermatitis (3, 4). Studies have shown that one potential physiological activator for hK7 is hK5, another member of the human tissue Kallikrein family. Along with hK14, these three kallikreins form a proteolytic cascade in the stratum corneum (5). The purified, secreted recombinant human K7 corresponds to the pro form. When activated by thermolysin, it displays enzymatic activity towards a fluorogenic synthetic peptide described in the Activity Assay Protocol. This activity can be inhibited by recombinant human Serpin A1, A3, A4, and A5 (Catalog # 1268-PI, 1295-PI, 1669-PI, and 1266-PI).

1. Hansson, L. et al. (1994) J. Bio. Chem. 269:19420.
2. Caubet, C. et al. (2004) J. Invest. Dermatol. 122:1235.
3. Ekholm, E. and Egelrud, T. (1999) Arch. Dermatol. Res. 291:195.
4. Hansson, L. et al. (2002) J. Invest. Dermatol. 118:444.
5. Brattsand, M. et al. (2004) J. Invest. Dermatol. 124:198.

Publications for Kallikrein 7 (2624-SE)(9)

Publications using 2624-SE

• K Kidana, T Tatebe, K Ito, N Hara, A Kakita, T Saito, S Takatori, Y Ouchi, T Ikeuchi, M Makino, TC Saido, M Akishita, T Iwatsubo, Y Hori, T Tomita Loss of kallikrein-related peptidase 7 exacerbates amyloid pathology in Alzheimer&#039;s disease model mice EMBO Mol Med, 2018;0(0):. 2018 [PMID: 29311134] (Bioassay, Human)
• N Fortelny, GS Butler, CM Overall, P Pavlidis Protease-inhibitor interaction predictions: Lessons on the complexity of protein-protein interactions Mol. Cell Proteomics, 2017;0(0):. 2017 [PMID: 28385878] (Bioassay)
• Yamamoto-Tanaka M, Motoyama A, Miyai M, Matsunaga Y, Matsuda J, Tsuboi R, Hibino T Mesotrypsin and caspase-14 participate in prosaposin processing: potential relevance to epidermal permeability barrier formation. J Biol Chem, 2014;289(29):20026-38. 2014 [PMID: 24872419] (Bioassay, Human)
• Fortugno P, Bresciani A, Paolini C, Pazzagli C, El Hachem M, D'Alessio M, Zambruno G Proteolytic activation cascade of the Netherton syndrome-defective protein, LEKTI, in the epidermis: implications for skin homeostasis. J Invest Dermatol, 2011;131(11):2223-32. 2011 [PMID: 21697885] (Enzyme Assay, Human)
• Kantyka T, Fischer J, Wu Z, Declercq W, Reiss K, Schroder J, Meyer-Hoffert U Inhibition of kallikrein-related peptidases by the serine protease inhibitor of Kazal-type 6. Peptides, 0;32(6):1187-92. 0 [PMID: 21439340] (Bioassay)
• Meyer-Hoffert U, Wu Z, Kantyka T, Fischer J, Latendorf T, Hansmann B, Bartels J, He Y, Glaser R, Schroder JM Isolation of SPINK6 in human skin: selective inhibitor of kallikrein-related peptidases. J. Biol. Chem., 2010;285(42):32174-81. 2010 [PMID: 20667819] (Enzyme Assay, Human)
• Sales KU, Masedunskas A, Bey AL Matriptase initiates activation of epidermal pro-kallikrein and disease onset in a mouse model of Netherton syndrome. Nat. Genet., 2010;42(8):676-83. 2010 [PMID: 20657595] (Bioassay, Human)
• Morizane S, Yamasaki K, Kabigting FD, Gallo RL Kallikrein expression and cathelicidin processing are independently controlled in keratinocytes by calcium, vitamin D(3), and retinoic acid. J. Invest. Dermatol., 2010;130(5):1297-306. 2010 [PMID: 20090765] (ELISA (Standard), N/A)
• Sexton DJ, Chen T, Martik D, Kuzmic P, Kuang G, Chen J, Nixon AE, Zuraw BL, Forteza RM, Abraham WM, Wood CR Specific inhibition of tissue kallikrein 1 with a human monoclonal antibody reveals a potential role in airway diseases. Biochem. J., 2009;422(2):383-92. 2009 [PMID: 19527222] (Surface Plasmon Resonance, Human)

Bioinformatics

• Gene Symbol: KLK7
• Uniprot:
  • P49862()