>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
<1.0 EU per 1 μg of the protein by the LAL method.
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.
1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
Substrate: BOC-Val-Pro-Arg-AMC (Catalog # ES011), 90 mM Stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
Dilute rhKLK4 to 200 µg/mL in Activation Buffer.
Dilute Thermolysin to 2 µg/mL in Activation Buffer.
Combine equal volumes diluted rhKLK4 and Thermolysin and incubate at 37 °C for 2 hours to activate.
Stop the reaction with 1,10 Phenanthroline at a final concentration of 10 mM.
Dilute activated rhKLK4 to 2 ng/µL in Assay Buffer.
Dilute Substrate to 200 µM in Assay Buffer.
Load 50 µL of the 2 ng/μL rhKLK4 in a plate and start the reaction by adding 50 μL of 200 μM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 200 µM Substrate without any rhKLK4.
Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), 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 7-Amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).
rhKLK4: 0.100 µg
Substrate: 100 µM
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Kallikrein 4/Prostase Protein, CF
Enamel matrix serine proteinase 1
kallikrein 4 (prostase, enamel matrix, prostate)
Kallikrein-like protein 1
kallikrein-related peptidase 4
PRSS17enamel matrix serine protease 1
PSTSandrogen-regulated message 1
Serine protease 17
Kallikrein 4 (KLK4), also known as prostase or enamel matrix serine protease 1 (EMSP1), is a serine protease of the human tissue kallikrein gene family (1). Among normal tissues, human KLK4 is specifically expressed in the prostate (2). It is over-expressed in prostate cancer and this expression is regulated by hormones including androgens, esterogen and progesterone (3). rhKLK4 readily activates pro-KLK3/PSA and pro-urokinase type plasminogen activator (uPA), indicating it may initiate events involving PSA and uPA in either normal or abnormal processes (4). KLK4 may have additional roles such as functioning as one of the two major enamel proteases identified that process enamel matrix proteins (5). In addition to being a secreted enzyme, it is also a nuclear protein (3, 6). The deduced amino acid sequence of human KLK4 consists of a signal peptide, a short pro region and a mature/active enzyme. rhKLK4 can be activated in vitro by thermolysin, a zinc protease. The peptidase activity can be inhibited by AEBSF (R&D Systems, Catalog # EI001), a general serine protease inhibitor.
Yousef, G.M. and E.P. Diamandis (2001) Endocrine Rev. 22:184.
Nelson, P.S. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3114.
Xi, Z. et al. (2004) Cancer Res. 64:2365.
Takayama, T.K. et al. (2001) Biochemistry 40:15341.
Simmer, J.P. and J.C. Hu (2002) Connect Tissue Res. 43:441.
Ryu, O.H. et al. (2002) Eur. J. Oral. Sci. 110:358.
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