Recombinant Mouse Legumain/Asparaginyl Endopeptidase, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse Legumain/Asparaginyl Endopeptidase, CF Summary

• Details of Functionality: 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.
• Source: Mouse myeloma cell line, NS0-derived mouse Legumain/Asparaginyl Endopeptidase protein
  Val18-Tyr435, with an N-terminal 7-His tag
• Accession #: O89017
• N-terminal Sequence: His
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Lgmn
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 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.
• SDS-PAGE: 61 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Activation Buffer: 0.1 M NaOAc, 0.1 M NaCl, pH 4.5
  • Assay Buffer: 50 mM MES, 250 mM NaCl, pH 5.5
  • Recombinant Mouse Legumain/Asparaginyl Endopeptidase (rmLegumain) (Catalog # 2058-CY)
  • 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
  1. Dilute rmLegumain to 50 µg/mL in Activation Buffer.
  2. Incubate for 4 hours at 37 °C.
  3. Dilute rmLegumain to 2 ng/µL in Assay Buffer.
  4. Dilute Substrate to 200 µM in Assay Buffer.
  5. 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.
  6. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  7. 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).

  Per Well:
  • rmLegumain: 0.100 µg
  • Substrate: 100 µM

Notes

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

• AEP
• Asparaginyl Endopeptidase
• cysteine protease 1
• Legumain
• LGMN
• LGMN1
• Protease, cysteine 1
• protease, cysteine, 1 (legumain)
• PRSC1EC 3.4.22.34

Background

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). 

1. Chen, J.M. et al. (1998) Biochem. J. 335:111.
2. Shirahama-Noda, K. et al. (2003) J. Biol. Chem. 278:33194.
3. Liu, C. et al. (2003) Cancer Res. 63: 2957.
4. Li D.N. et al. (2003) J. Biol. Chem. 278:38980.
5. Chen, J.M. et al. (2001) Biol. Chem. 382:777.
6. Schwarz, G. et al. (2002) Biol. Chem. 383:1813.
7. Sarndeses, C.S. et al. (2003) J. Biol. Chem. 278:13286.

Publications for Legumain/Asparaginyl Endopeptidase (2058-CY)(2)

Publications using 2058-CY

• Lustenberger, SK;Castro Jaramillo, CA;Bärtschi, LA;Williams, R;Schibli, R;Mu, L;Krämer, SD; Towards imaging the immune state of cancer by PET: Targeting legumain with 11C-labeled P1-Asn peptidomimetics carrying a cyano-warhead Nuclear medicine and biology 2024-09-13 [PMID: 39303441] (Bioassay, N/A)
• Cheng, Q;Ma, X;Liu, J;Feng, X;Liu, Y;Wang, Y;Ni, W;Song, M; Pharmacological Inhibition of the Asparaginyl Endopeptidase (AEP) in an Alzheimer's Disease Model Improves the Survival and Efficacy of Transplanted Neural Stem Cells International journal of molecular sciences 2023-04-23 [PMID: 37175445] (Bioassay, Mouse)

Bioinformatics

• Gene Symbol: Lgmn
• Uniprot:
  • O89017()