• 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 a fluorogenic peptide substrate, Mca-SEVNLDAEFRK(Dpn)RR-NH    ₂ (Catalog # ). The specific activity is >350 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Leu21-Ser593 & Thr23-Ser593, both with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Leu21 & Thr23

• Structure / Form

  Pro form    
   

• Predicted Molecular Mass

  66 kDa

• SDS-PAGE

  85 kDa, reducing conditions

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 8.0

• Recombinant Human Meprin  beta  Subunit/MEP1B (rhMEP1B) (Catalog # 2895-ZN)

• Trypsin (Sigma, Catalog # T-1426 )

• AEBSF (Catalog # ), 100 mM stock in water

• Substrate: MCA-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Arg-Lys(DNP)-Arg-Arg-NH₂ (Catalog # )

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhMEP1B to 100 µg/mL with 1 µg/mL Trypsin in Activation Buffer.

2. Incubate for 45 minutes at 37 °C. Stop Trypsin activation by adding AEBSF at a final concentration of 1 mM in Activation Buffer and mixing well.

3. Dilute activated rhMEP1B to 0.4 µg/mL in Assay Buffer.

4. Dilute Substrate to 20 µM in Assay Buffer.

5. Load 50 µL of 0.4 µg/mL rhMEP1B into plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate.

6. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.

7. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

Per Well:

• rhMEP1B: 0.020 µg

• Substrate: 10 µM
  

Background: Meprin beta Subunit/MEP1B

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 Merpin A (composed of alpha subunits with or without beta subunits) and Merpin 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 701 amino acid sequence of human Merpin beta subunit precursor consists of a signal peptide (residues 1 to 21), a pro region (residues 22 to 61), and a mature chain (residues 62 to 701) containing the following domains, catalytic (residues 62 to 259), MAM (residues 260 to 429), MATH (residues 430 to 585), EGF-like (residues 604 to 644), transmembrane (residues 653 to 673), and cytoplasmic (residues 674 to 701). The pro enzyme terminating at residue 593 was expressed and the secreted protein purified from conditioned medium. After trypsin treatment, the activated enzyme cleaved a flurogenic peptide, which contains Asp and Glu, the preferred residues found in the P1’ and P1 sites (3).

• References:

1. Bond, J.S. and R.J. Beynon (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.

• Entrez Gene IDs:

  4225 (Human); 17288 (Mouse)

• Alternate Names:

  EC 3.4.24.63; endopeptidase-2; MEP1B; meprin A subunit beta; meprin A, beta; Meprin B; Meprin beta Subunit; N-benzoyl-L-tyrosyl-p-amino-benzoic acid hydrolase beta subunit; N-benzoyl-L-tyrosyl-P-amino-benzoic acid hydrolase subunit beta; PABA peptide hydrolase; PPH beta