Recombinant Human Kallikrein 3/PSA Protein, CF Summary
Details of Functionality
Measured by its ability to cleave the colorimetric peptide substrate, Succinyl-Arg-Pro-Tyr-p-Nitroanilide (Suc-RPY-pNA). The specific activity is >70 pmol/min/µg, as measured under the described conditions.
Mouse myeloma cell line, NS0-derived human Kallikrein 3/PSA protein Ala18-Pro261, with a C-terminal 10-His tag
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
<1.0 EU per 1 μg of the protein by the LAL method.
28 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: Suc-Arg-Pro-Tyr-pNa (AnaSpec, Catalog # 20586), 10 mM in deionized water
96-well Clear Plate (Costar, Catalog # 92592)
Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Dilute rhKLK3 to 200 µg/mL in Activation Buffer.
Dilute Thermolysin to 2 µg/mL in Activation Buffer.
Combine equal volumes of 200 µg/mL rhKLK3 and 2 µg/mL Thermolysin.
Incubate at 37 °C for 5 minutes.
Dilute 1,10 Phenanthroline to 20 mM in Assay Buffer.
Stop Thermolysin activity by adding 1,10 Phenanthroline to a final concentration of 10 mM.
Dilute activated rhKLK3 to 20 ng/µL in Assay Buffer.
Dilute Substrate to 2 mM in Assay Buffer.
Load 50 µL of the 20 ng/µL rhKLK3 in a plate and start the reaction by adding 50 µL of 2 mM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 2 mM Substrate.
Read at a wavelength of 405 nm (bottom read) in kinetic mode for 5 minutes.
Calculate specific activity:
Specific Activity (pmol/min/µg) =
Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol
ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)
*Adjusted for Substrate Blank **Using the extinction coefficient 8800 M-1cm-1 ***Using the path correction 0.32 cm Note: the output of many spectrophotometers is in mOD. Per Well:
rhKLK3: 1 µg
Substrate: 1 mM
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Kallikrein 3/PSA Protein, CF
kallikrein 3, (prostate specific antigen)
kallikrein-related peptidase 3
prostate specific antigen
Kallikrein 3, commonly known as prostate specific antigen (PSA), is a serine protease of the human tissue Kallikrein gene family (1). PSA is synthesized in the ductal and acinar epithelium of the prostate gland and secreted into the seminal plasma in high concentrations (0.5 - 2 g/L) (2). A small portion of PSA “leaks” into the systemic circulation, the levels of which increase significantly (30-fold) from prostate cancer tissue than normal prostate tissue (3). PSA has become a well established tumor marker that aids the diagnosis, staging, and follow up of prostate cancer.
The deduced amino acid sequence of human PSA consists of a signal peptide, a short pro region and a mature/active enzyme. The pro-enzyme is activated, possibly by active Kallikreins 2, 4 or 15 in vivo (4). rhPSA is activated by thermolysin, a zinc protease. The active PSA cleaves several tyrosyl peptide bonds in semenogelins I and II, which are the major gel-forming proteins produced by the seminal vesicles (5). Several inhibitors including serpin A3/ alpha 1-antichymotrypsin (ACT) and alpha 2‑macroglobulin are known to form complexes with PSA.
Yousef, G.M. and E.P. Diamandis (2001) Endocrine Rev. 22:184.
Ward, A.M. et al. (2001) Ann. Clin. Biochem. 38:633.
Jain, S. et al. (2002) Postgrad. Med. J. 78:646.
LiLja H. (2003) Urology 62:27.
Takayama, T.K. et al. (1997) J. Biol. Chem. 272:21582.
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