Recombinant Human Kallikrein 1 Protein, CF Summary
Details of Functionality
Measured by its ability to cleave a flourogenic peptide substrate Pro-Phe-Arg-7-amido-4-methylcoumarin (PFR-AMC). The specific activity is >1,500 pmol/min/µg, as measured under the described conditions.
Mouse myeloma cell line, NS0-derived human Kallikrein 1 protein Ile25-Ser262, with an N-terminal human KLK-5 propeptide (Val23-Arg66) and a C-terminal 10-His tag
>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.
32 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: Pro-Phe-Arg-AMC (Bachem, Catalog # I-1295), 10 mM stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
Dilute rhKLK1 to 200 µg/mL in Activation Buffer.
Dilute Thermolysin to 2 µg/mL in Activation Buffer.
Combine 20 µL of diluted rhKLK1 with 20 µL of diluted Thermolysin for a final concentrations of 100 µg/mL and 1 µg/mL respectively.
Incubate at 37 °C for 1 hour.
Stop the reaction with 40 µL of 20 mM 1,10 Phenanthroline for a final concentration of 10 mM.
Dilute incubated rhKLK1 to 0.4 ng/µL in Assay Buffer.
Dilute Substrate to 200 µM in Assay Buffer.
Load 50 µL of the 0.4 ng/µL rhKLK1 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.
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).
rhKLK1: 0.02 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 1 Protein, CF
glandular kallikrein 1
kallikrein 1, renal/pancreas/salivary
kallikrein serine protease 1
Kidney/pancreas/salivary gland kallikrein
Kallikrein 1 (KLK1), also known as tissue kallikrein, is a member of human tissue kallikrein family (1). The best known physiological function of KLK1 is the cleavage of kininogen to release the vasoactive kinin peptide (bradykinin or lysyl-bradykinin), which regulates vasodilation, blood pressure reduction, smooth muscle relaxation and contraction, pain induction and inflammation (2). In addition, KLK1 may play a role in angiogensis and tumorigenesis (2). Human KLK1 precursor contains a signal peptide (residues1 to 18), a short pro peptide (residues 19 to 24) and a mature chain (residues 25 to 262). The purified rhKLK1 contains the mature chain and the propeptide from human KLK5. After being activated by thermolysin, rhKLK1 is active against a fluorogenic peptide substrate described above.
Yousef, G.M. and E.P. Diamandis (2001) Endocrine Rev. 22:184.
Chao, J. (2004) in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. eds. pp. 1577 - 1580.
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