Recombinant Human Legumain/Asparaginyl Endopeptidase, CF

Catalog # Availability Size / Price Qty
2199-CY-010
R&D Systems Recombinant Proteins and Enzymes
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Product Details
Citations (7)
FAQs
Reviews (2)

Recombinant Human Legumain/Asparaginyl Endopeptidase, CF Summary

Product Specifications

Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
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 >250 pmol/min/µg, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived human Legumain/Asparaginyl Endopeptidase protein
Ile18-Tyr433, with an N-terminal 7-His tag
Accession #
N-terminal Sequence
Analysis
His
Structure / Form
Pro form
Predicted Molecular Mass
49 kDa
SDS-PAGE
60 kDa, reducing conditions

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2199-CY

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

2199-CY

Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  • Activation Buffer: 50 mM Sodium Acetate, 100 mM NaCl, pH 4.0
  • Assay Buffer: 50 mM MES, 250 mM NaCl, pH 5.0
  • Recombinant Human Legumain/Asparaginyl Endopeptidase (rhLegumain) (Catalog # 2199-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 rhLegumain to 100 µg/mL in Activation Buffer.
  2. Incubate for 2 hours at 37 °C.
  3. Dilute rhLegumain to 1 ng/µL in Assay Buffer.
  4. Dilute Substrate to 200 µM in Assay Buffer.
  5. Load into a black well plate 50 µL of 1 ng/µL rhLegumain 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.
  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:
  • rhLegumain: 0.050 µg
  • Substrate: 100 µM
Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Legumain/Asparaginyl Endopeptidase

Legumain is a lysosomal cysteine protease whose activity is found in several tissues tested (1, 2). 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 (3). Over-expression of Legumain in tumors is significant for invasion/metastasis (4). 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 the cleavage, which result in the removal of the pro peptides in both C- and N-termini (5). 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 (6‑8). Human Legumain is synthesized as a 433 amino acid precursor with a signal peptide (residues 1‑17). The pro enzyme (residues 18‑433) was expressed with an N-terminal His tag. This activity of Legumain can be inhibited by recombinant human Cystatins C and E/M and recombinant mouse Cystatin C (Catalog # 1196-PI1286-PI and 1238-PI, respectively).

References
  1. Chen, J.-M. et al. (1997) J. Biol. Chem. 272:8090.
  2. Tanaka, T. et al. (1996) Cytogenet. Cell Genet. 74:120.
  3. Shirahama-Noda, K. et al. (2003) J. Biol. Chem. 278:33194.
  4. Liu, C. et al. (2003) Cancer Res. 63: 2957.
  5. Li D.N. et al. (2003) J. Biol. Chem. 278:38980.
  6. Chen, J.M. et al. (2001) Biol. Chem. 382:777.
  7. Schwarz, G. et al. (2002) Biol. Chem. 383:1813.
  8. Sarndeses, C.S. et al. (2003) J. Biol. Chem. 278:13286.
Entrez Gene IDs
5641 (Human); 19141 (Mouse)
Alternate Names
AEP; Asparaginyl Endopeptidase; cysteine protease 1; Legumain; LGMN; LGMN1; Protease, cysteine 1; protease, cysteine, 1 (legumain); PRSC1EC 3.4.22.34

Citations for Recombinant Human Legumain/Asparaginyl Endopeptidase, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

7 Citations: Showing 1 - 7
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  1. An endogenous stimulus detonated nanocluster-bomb for contrast-enhanced cancer imaging and combination therapy
    Authors: H Sun, W Ma, S Duan, J Huang, R Jia, H Cheng, B Chen, X He, K Wang
    Chemical Science, 2021;12(36):12118-12129.
    Applications: Enzyme Assay
  2. N368-Tau fragments generated by legumain are detected only in trace amount in the insoluble Tau aggregates isolated from AD brain
    Authors: K Schlegel, K Awwad, RG Heym, D Holzinger, A Doell, S Barghorn, TR Jahn, C Klein, Y Mordashova, M Schulz, L Gasparini
    Acta Neuropathol Commun, 2019;7(1):177.
    Species: N/A
    Sample Types: Recombinant Protein
    Applications: Enzyme Assay
  3. Structural and functional characterization of the triticale (x Triticosecale Wittm.) phytocystatin TrcC-8 and its dimerization-dependent inhibitory activity
    Authors: B Prabucka, M Mielecki, M Chojnacka, W Bielawski, M Czarnocki-, S Orzechowsk
    Phytochemistry, 2017;142(0):1-10.
    Species: N/A
    Sample Types: Recombinant Protein
    Applications: Bioassay
  4. Insights into the mechanism of cystatin C oligomer and amyloid formation and its interaction with beta-amyloid
    Authors: TJ Perlenfein, JD Mehlhoff, RM Murphy
    J. Biol. Chem., 2017;0(0):.
    Species: Human
    Sample Types: Protein
    Applications: Bioassay
  5. Structural Basis for the Function of Complement Component C4 within the Classical and Lectin Pathways of Complement.
    Authors: Mortensen S, Kidmose R, Petersen S, Szilagyi A, Prohaszka Z, Andersen G
    J Immunol, 2015;194(11):5488-96.
    Species: Human
    Sample Types: Protein
    Applications: Enzyme Assay
  6. Cystatin E/M suppresses legumain activity and invasion of human melanoma.
    Authors: Briggs JJ, Haugen MH, Johansen HT, Riker AI, Abrahamson M, Fodstad O, Maelandsmo GM, Solberg R
    BMC Cancer, 2010;10(1):17.
    Species: N/A
    Sample Types: N/A
    Applications: Western Blot
  7. Expression of the cysteine protease legumain in vascular lesions and functional implications in atherogenesis.
    Authors: Clerin V, Shih HH, Deng N, Hebert G, Resmini C, Shields KM, Feldman JL, Winkler A, Albert L, Maganti V, Wong A, Paulsen JE, Keith JC, Vlasuk GP, Pittman DD
    Atherosclerosis, 2008;0(0):.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay

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Reviews for Recombinant Human Legumain/Asparaginyl Endopeptidase, CF

Average Rating: 5 (Based on 2 Reviews)

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Recombinant Human Legumain/Asparaginyl Endopeptidase, CF
By Anonymous on 12/04/2020
Application: In vitro bioactivity in cell culture

Recombinant Human Legumain/Asparaginyl Endopeptidase, CF
By Dane Jensen on 12/04/2020
Application: In vitro bioactivity in cell culture
Reason for Rating: Legumain was consistent from vial to vial throughout the experiments. The study lasted over a year.