Measured by its ability to cleave the fluorogenic peptide substrate, N-carbobenzyloxy-Ala-Ala-Asn-7-amido-4-methylcoumarin (Z-AAN-AMC). The specific activity is >350 pmol/min/µg, as measured under the described conditions.
Mouse myeloma cell line, NS0-derived mouse Legumain/Asparaginyl Endopeptidase protein Val18-Tyr435, with an N-terminal 7-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.
49 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.
61 kDa, reducing conditions
Read Publication using 2058-CY in the following applications:
Substrate: Z-Ala-Ala-Asn-AMC (Bachem, Catalog # I-1865), 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 rmLegumain to 50 µg/mL in Activation Buffer.
Incubate for 4 hours at 37 °C.
Dilute rmLegumain to 2 ng/µL in Assay Buffer.
Dilute Substrate to 200 µM in Assay Buffer.
Load 50 µL of 2 ng/µL rmLegumain in the plate, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing Assay Buffer and 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 (AMC) (Sigma, Catalog # A-9891).
rmLegumain: 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 Mouse Legumain/Asparaginyl Endopeptidase, CF
cysteine protease 1
Protease, cysteine 1
protease, cysteine, 1 (legumain)
Legumain is a lysosomal cysteine protease found in all mouse tissues examined, but was particularly abundant in kidney and placenta (1). Legumain plays a pivotal role in the endosomal/lysosomal degradation system because the Legumain deficiency causes the accumulation of pro cathepsins B, H and L, another group of lysosomal cysteine proteases (2). Over-expression of Legumain in tumors is significant for invasion/metastasis (3). Also known as asparaginyl endopeptidase, it specifically cleaves peptide bonds with Asn at the P1 position. Nevertheless, it also cleaves peptide bonds with Asp at the P1 position. Auto-activation of pro Legumain involves both types of cleavage, which results in the removal of the pro peptides in both C- and N-termini (4). In addition, Legumain activates pro MMP-2 and processes bacterial antigens for MHC class II presentation and pro thymosin alpha to thymosin alpha 1 and thymosin alpha 11, two acidic peptides with immunoregulatory properties (5-7). Mouse Legumain is synthesized as a 435 amino acid precursor with a signal peptide (residues 1 to 17). The pro enzyme (residues 18 to 435) was expressed with an N-terminal His tag. The purified pro enzyme can be activated under the conditions as described above. Legumain activity can be inhibited by rmCystatin C and recombinant human cystatins C and E/M (Catalog # 1238-PI, 1196-PI, and 1286-PI).
Chen, J.M. et al. (1998) Biochem. J. 335:111.
Shirahama-Noda, K. et al. (2003) J. Biol. Chem. 278:33194.
Liu, C. et al. (2003) Cancer Res. 63: 2957.
Li D.N. et al. (2003) J. Biol. Chem. 278:38980.
Chen, J.M. et al. (2001) Biol. Chem. 382:777.
Schwarz, G. et al. (2002) Biol. Chem. 383:1813.
Sarndeses, C.S. et al. (2003) J. Biol. Chem. 278:13286.
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