Recombinant Human Meprin alpha Subunit/MEP1A Protein, CF

Catalog # Availability Size / Price Qty
3220-ZN-010
Product Details
Citations (3)
FAQs
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Recombinant Human Meprin alpha Subunit/MEP1A Protein, CF Summary

Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to cleave a fluorogenic peptide substrate, Mca-YVADAPK(Dnp)-OH (Catalog # ES007). The specific activity is >500 pmol/min/µg, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived human Meprin alpha Subunit/MEP1A protein
Val22-Gln601 & Leu27-Gln601, both with a C-terminal 10-His tag
Accession #
N-terminal Sequence
Analysis
Val22 and Leu27
Structure / Form
Disulfide-linked homodimer
Predicted Molecular Mass
67 kDa
SDS-PAGE
86 kDa, reducing conditions

Product Datasheets

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.

3220-ZN

Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Shipping The product is shipped with polar packs. 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: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Assay Buffer: 50 mM Tris, pH 7.5
  • Recombinant Human Meprin  alpha  Subunit/MEP1A (rhMEP1A) (Catalog # 3220-ZN)
  • Activator: Trypsin (Sigma, Catalog # T-1426)
  • AEBSF (Tocris, Catalog # 5175), 100 mM stock in deionized water
  • Fluorogenic Peptide Substrate: MCA-Tyr-Val-Ala-Asp-Ala-Pro-Lys(DNP)-OH (Catalog # ES007)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhMEP1A to 100 µg/mL in Activation Buffer with 0.1 µg/mL Trypsin.
  2. Incubate 100 µg/mL rhMEP-1A at 37 °C for 3 hours.
  3. Stop Trypsin activity by adding AEBSF to a final concentration of 1 mM.
  4. Dilute activated rhMEP1A to 0.4 µg/mL in Assay Buffer.
  5. Dilute Substrate to 40 µM in Assay Buffer.
  6. Load into a black well plate 50 µL of 0.4 µg/mL rhMEP1A, and start the reaction by adding 50 µL of 40 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 40 µM Substrate.
  7. Read excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  8. 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 MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

Per Well:
  • rhMEP1A: 0.02 µg
  • Substrate: 20 µM
Reconstitution Calculator

Reconstitution Calculator

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Background: Meprin alpha Subunit/MEP1A

Meprins are multimeric proteases composed of alpha and beta subunits, which are members of the astacin family of zinc endopeptidases (1, 2). Both subunits form disulfide-linked homo- or heterooligomers, which are also referred to as Meprin A (composed of alpha subunits with or without beta  subunits) and Meprin B (composed of beta subunits only) (3). Although the two subunits share 42% identity in their amino acid sequence, they differ significantly in their oligomeric structure, post-translational processing and subsequently cellular location, and substrate and peptide bond specificity (4). The 746 amino acid sequence of human meprin alpha subunit precursor consists of a signal peptide (residues 1 to 21), a pro region (residues 22 to 65), and a mature chain (residues 66 to 746) containing following domains, catalytic (residues 62 to 263), MAM (residues 264 to 433), MATH (residues 434 to 593), EGF-like (residues 670 to 710), transmembrane (residues 713 to 740), and cytoplasmic (residues 741 to 746). The pro enzyme terminating at residue 601 was expressed and the secreted protein purified from conditioned medium. The molecular masses of recombinant human MEP1A are similar to those observed for the alpha subunit of rat Meprin A (5).

References
  1. Bond, J.S. and Beynon, R.J. (1995) Protein Sci. 4:1247.
  2. Stocker, W. et al. (1995) Protein Sci. 4:823.
  3. Bertenshaw, G.P., et al. (2001) J. Biol. Chem. 276:13248.
  4. Ishmael, F.T. et al. (2005) J. Biol. Chem. 280:13895.
  5. Bertenshaw, G.P., et al. (2003) J. Biol. Chem. 278:2522.
Entrez Gene IDs
4224 (Human); 17287 (Mouse); 25684 (Rat)
Alternate Names
bA268F1.1 (meprin A alpha (PABA peptide hydrolase)); EC 3.4.24; EC 3.4.24.18; endopeptidase-2; MEP1A; meprin A subunit alpha; meprin A, alpha (PABA peptide hydrolase); Meprin alpha Subunit; N-benzoyl-L-tyrosyl-P-amino-benzoic acid hydrolase subunit alpha; PABA peptide hydrolase; PPH alpha; PPHA

Citations for Recombinant Human Meprin alpha Subunit/MEP1A Protein, 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.

3 Citations: Showing 1 - 3
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  1. Meprin ? contributes to collagen deposition in lung fibrosis
    Authors: V Biasin, M Wygrecka, LM Marsh, C Becker-Pau, L Brcic, B Ghanim, W Klepetko, A Olschewski, G Kwapiszews
    Sci Rep, 2017;7(0):39969.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  2. The ovicidal, larvacidal and adulticidal properties of 5,5'-dimethyl-2,2'-bipyridyl against Drosophila melanogaster.
    Authors: Van Hiel, Matthias, Breugelmans, Bert, Pagel, Charles, Williams, Adam K, Varan, Aiden K, Burke, Richard, Bowles, Vernon M, Batterham, Philip
    PLoS ONE, 2012;7(11):e49961.
    Species: N/A
    Sample Types: N/A
    Applications: Zymography
  3. Astacin proteases cleave dentin sialophosphoprotein (Dspp) to generate dentin phosphoprotein (Dpp).
    Authors: Tsuchiya S, Simmer JP, Hu JC, Richardson AS, Yamakoshi F, Yamakoshi Y
    J. Bone Miner. Res., 2010;26(0):220.
    Species: Human
    Sample Types:
    Applications: Bioassay

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Fluorogenic Peptide Substrates

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