Recombinant Mouse Legumain/Asparaginyl Endopeptidase, CF

Catalog #: 2058-CY
2 Citations Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
2058-CY-010

R&D Systems Recombinant Proteins and Enzymes

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All Legumain/Asparaginyl Endopeptidase Proteins and Enzymes

Product Details

Citations (2)

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Mouse 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 >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
Analysis

His

Structure / Form

Pro form

Predicted Molecular Mass

49 kDa

SDS-PAGE

61 kDa, reducing conditions

Product Datasheets

2058-CY

Product Datasheet

COA

SDS

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.

2058-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, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials

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

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 V_max* (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

Reconstitution Calculator

Background: Legumain/Asparaginyl Endopeptidase

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).

References

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.

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

Related Research Areas

Citations for Recombinant Mouse 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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