Recombinant Human Kallikrein 11 Protein, CF Summary
Details of Functionality
Measured by its ability to cleave a colorimetric peptide substrate D-Val-Leu-Lys-ThioBenzyl ester (VLK-SBzl), in the presence of 5,5’Dithio-bis (2-nitrobenzoic acid) (DTNB). Edwards, K.M. et al. (1999) J. Biol. Chem. 274:30468. The specific activity is >200 pmol/min/µg, as measured under the described conditions.
Mouse myeloma cell line, NS0-derived human Kallikrein 11 protein Glu19-Asn250, with an N-terminal signal peptide and 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.
27 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.
Fluorogenic Peptide Substrate: Z-Lys-SBzl (Bachem, Catalog # M-1300), 10 mM stock in DMSO
5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D-8130), 10 mM stock in DMSO
96-well Clear Plate (Costar, Catalog # 92592)
Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Dilute rhKLK11 to 200 µg/mL in Activation Buffer.
Dilute Thermolysin to 20 μg/mL in Activation Buffer.
Combine equal volumes of 200 µg/mL rhKLK11 and 20 µg/mL Thermolysin.
Incubate at 37 °C for 15 minutes.
Stop Thermolysin activity by adding EDTA to a final concentration of 10 mM.
Dilute incubated rhKLK11 to 2 ng/µL in Assay Buffer.
Prepare Substrate Mixture containing 600 μM Substrate and 200 μM DTNB in Assay Buffer.
Load into a clear plate 50 µL of the 2 ng/µL rhKLK11, and start the reaction by adding 50 µL of Substrate Mixture. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of Substrate Mixture.
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 13260 M-1cm-1 ***Using the path correction 0.320 cm Note: the output of many spectrophotometers is in mOD Per Well:
rhKLK11: 0.1 µg
Substrate: 300 µM
DTNB: 100 µM
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Kallikrein 11 Protein, CF
kallikrein-related peptidase 11
Serine protease 20
As a member of human tissue kallikrein family, Kallikrein 11, also known as hippostasin, trypsin-like serine protease and PRSS20, is encoded by the KLK11 gene (1). Two alternatively spliced forms exist, resulting in 250 (isoform 1) and 282 (isoform 2) amino acid sequences,respectively (2-5). Isoform 1 consists of a signal peptide (residues 1-18), a short pro peptide (residues 19-21) and the mature chain (residues 22-250). Isoform 2 is identical to isoform 1, except that a 32 amino acid segment is inserted in isoform 2 before residue 1 in isoform 1. Isoform 1 is predominantly expressed in brain whereas isoform 2 is preferentially expressed in prostate. KLK11 is a novel marker for ovarian and prostate cancer carcinomas (6-8). Recombinant human KLK11, after being activated by thermolysin, is active against a thioester substrate. This activity can be inhibited by AEBSF (Catalog # EI001), dichloroisocoumarin, and aprotinin. Recombinant human KLK11 produced by R&D Systems corresponds to isoform 1.
Yousef, G.M. and E.P. Diamandis (2001) Endocrine Rev. 22:184.
Yoshida, S. et al. (1998) Biochim. Biophys. Acta 1399:225.
Yousef, G.M. et al. (2000) Genomics 63:88.
Mitsui, S. et al. (2000) Biochem. Biophys. Res. Commun. 272:205.
Gan, L. et al. (2000) Gene 257:119.
Diamandis, E.P. et al. (2002) Cancer Res. 62:295.
Nakamura, T. et al. (2003) Urology 61:1042.
Borgono, C.A. et al. (2003) Int. J. Cancer 106:605.
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