Recombinant Human Legumain/Asparaginyl Endopeptidase, CF
Recombinant Human Legumain/Asparaginyl Endopeptidase, CF Summary
Product Specifications
Ile18-Tyr433, with an N-terminal 7-His tag
Analysis
Product Datasheets
Carrier Free
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.
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.
|
Assay Procedure
- 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
- Dilute rhLegumain to 100 µg/mL in Activation Buffer.
- Incubate for 2 hours at 37 °C.
- Dilute rhLegumain to 1 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- 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.
- 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).
Per Well:- rhLegumain: 0.050 µg
- Substrate: 100 µM
Reconstitution Calculator
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-PI, 1286-PI and 1238-PI, respectively).
- Chen, J.-M. et al. (1997) J. Biol. Chem. 272:8090.
- Tanaka, T. et al. (1996) Cytogenet. Cell Genet. 74:120.
- 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.
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.
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Citations: Showing 1 - 8
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An evolutionary molecular adaptation of an unusual stefin from the liver fluke Fasciola hepatica redefines the cystatin superfamily
Authors: M Buša, Z Matoušková, P Bartošová-, P Pachl, P ?ezá?ová, RM Eichenberg, P Deplazes, M Horn, S Štefani?, M Mareš
The Journal of Biological Chemistry, 2023-02-01;0(0):102970.
Species: N/A
Sample Types: Recombinant Protein
Applications: Bioassay -
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, 2021-08-17;12(36):12118-12129.
Applications: Enzyme Assay -
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, 2019-11-13;7(1):177.
Species: N/A
Sample Types: Recombinant Protein
Applications: Enzyme Assay -
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, 2017-06-24;142(0):1-10.
Applications: Bioassay -
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., 2017-05-09;0(0):.
Species: Human
Sample Types: Protein
Applications: Bioassay -
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, 2015-04-24;194(11):5488-96.
Species: Human
Sample Types: Protein
Applications: Enzyme Assay -
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, 2010-01-15;10(1):17.
Applications: Western Blot -
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, 2008-02-21;0(0):.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay
FAQs
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View all Proteins and Enzyme FAQsReviews for Recombinant Human Legumain/Asparaginyl Endopeptidase, CF
Average Rating: 5 (Based on 2 Reviews)
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Reason for Rating: Legumain was consistent from vial to vial throughout the experiments. The study lasted over a year.
