Recombinant Human Kallikrein 6 Protein, CF

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

Recombinant Human Kallikrein 6 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate Boc-QAR-AMC (Catalog # ES014). The specific activity is >1300 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Kallikrein 6/Neurosin protein
  Glu17-Lys244, with a C-terminal 10-His tag
• Accession #: Q92876
• N-terminal Sequence: Glu17
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: KLK6
• 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: 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: 35 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 Sodium Citrate.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • Activation Buffer: 50 mM Tris, 0.05% (w/v) Brij-35, pH 8.0
  • Assay Buffer: 50 mM Tris, 1.0 M Sodium Citrate, pH 7.5
  • Recombinant Human Kallikrein 6/Neurosin (rhKLK6) (Catalog # 5164-SE)
  • Lysyl-Endopeptidase, 20 U/mL stock in PBS
  • Substrate: BOC-Gln-Ala-Arg-AMC (Catalog # ES014), 10 mM stock in DMSO
  • Black 96-well Plate
  • Plate Reader with Fluorescence Read Capability
  1. Dilute rhKLK6 to 200 µg/mL in Activation Buffer.
  2. Dilute Lysyl-endopeptidase to 2.5 mU/mL in Activation Buffer.
  3. Combine and mix equal volumes of diluted rhKLK6 and diluted Lysyl-endopeptidase.
  4. Incubate at room temperature for 30 minutes.
  5. After incubation, dilute rhKLK6 to 0.5 µg/mL in Assay Buffer.
  6. Dilute Substrate to 200 µM in Assay Buffer.
  7. Load into a black well plate 50 µL of 0.5 µg/mL rhKLK6, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 200 µM Substrate.
  8. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  9. 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 7-Amino, 4-Methyl Coumarin (AMC).

  Per Well:
  • rhKLK6: 0.025 µg
  • Substrate: 100 µM

Notes

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

Alternate Names for Recombinant Human Kallikrein 6 Protein, CF

• Bssp
• EC 3.4.21
• EC 3.4.21.-
• hK6
• kallikrein 6 (neurosin, zyme)
• Kallikrein 6
• kallikrein-6
• kallikrein-related peptidase 6
• KLK6
• Klk7
• Neurosin
• Protease M
• protease, serine, 18
• PRSS18
• PRSS9
• PRSS9MGC9355
• Serine protease 18
• Serine protease 9
• SP59
• Zyme

Background

Tissue kallikreins are a family of extracellular serine proteases consisting of 15 members (1, 2). The genes of this family aligned in tandem on chromosome 19q13.4 represent the largest contiguous group of proteases within the human genome. Tissue kallikreins have attracted great interest as potential biomarkers for various cancers, including prostate, ovarian, breast, testicular, and lung (2, 3). Human Kallikrein 6 (hKLK6) is a member of tissue kallikrein family observed in breast and brain tissues, colon carcinoma cells, and oligodedrocytes (1 - 3). Known protein substrates of hKLK6 are myelin basic protein, the precursor of the A beta amyloid peptide, and plasminogen. Its physiological functions may include the participation in demyelination processes as well as in the progression of inflammatory disease of the CNS. The level of hKLK6 is reduced in brain extracts of Alzheimer patients and increased in serum of patients with ovarian cancer.

1. Emami, N. and E.P. Diamandis (2007) Clin Chim Acta 381:78.
2. Borgoño, C.A. and E.P. Diamandis (2004) Nat. Rev. Cancer, 4:876.
3. Paliouras, M. et al. (2007) Cancer Lett. 249:61.

Publications for Kallikrein 6/Neurosin (5164-SE)(7)

Publications using 5164-SE

• E Bosc, J Anastasie, F Soualmia, P Coric, JY Kim, LQ Wang, G Lacin, K Zhao, R Patel, E Duplus, P Tixador, AA Sproul, B Brugg, M Reboud-Rav, CM Troy, ML Shelanski, S Bouaziz, M Karin, C El Amri, ED Jacotot Genuine selective caspase-2 inhibition with new irreversible small peptidomimetics Cell Death & Disease, 2022-11-15;13(11):959. 2022-11-15 [PMID: 36379916] (Bioassay, N/A)
• H Bouzid, F Soualmia, K Oikonomopo, A Soosaipill, F Walker, K Louati, R Lo Dico, M Pocard, C El Amri, NA Ignatenko, D Darmoul Kallikrein-Related Peptidase 6 (KLK6) as a Contributor toward an Aggressive Cancer Cell Phenotype: A Potential Role in Colon Cancer Peritoneal Metastasis Oncogene, 2022-07-19;12(7):. 2022-07-19 [PMID: 35883559] (Bioassay, Human)
• 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, 2018-03-01;0(0):. 2018-03-01 [PMID: 29311134] (Bioassay, Human)
• Wennstrom M, Surova Y, Hall S, Nilsson C, Minthon L, Bostrom F, Hansson O, Nielsen H Low CSF Levels of Both alpha-Synuclein and the alpha-Synuclein Cleaving Enzyme Neurosin in Patients with Synucleinopathy. PLoS ONE, 2013-01-08;8(1):e53250. 2013-01-08 [PMID: 23308173] (ELISA (Standard), Western Blot)
• Spencer B, Michael S, Shen J, Kosberg K, Rockenstein E, Patrick C, Adame A, Masliah E Lentivirus mediated delivery of neurosin promotes clearance of wild-type alpha-synuclein and reduces the pathology in an alpha-synuclein model of LBD. Mol Ther, 2012-04-17;21(1):31-41. 2012-04-17 [PMID: 22508489] (Bioassay)
• 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, 2011-03-23;32(6):1187-92. 2011-03-23 [PMID: 21439340] (Bioassay)
• 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., 2009-08-13;422(2):383-92. 2009-08-13 [PMID: 19527222] (Surface Plasmon Resonance, Human)

Bioinformatics

• Gene Symbol: KLK6
• Uniprot:
  • Q92876()