• Purity

  >75%, 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 Boc-QAR-AMC (Catalog # ). The specific activity is >4,000 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  E. coli-derived Gly596-Val855, with an N-terminal Met and 6-His tag The protein was auto-activated and further purified.    
   

• Accession #

• N-terminal Sequence    
  Analysis

  Met & Val615

• Predicted Molecular Mass

  3 kDa & 26 kDa

• SDS-PAGE

  28 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM Tris, 50 mM NaCl, 0.01% (v/v) Tween® 20, pH 9.0

• Recombinant Mouse Matriptase/ST14 Catalytic Domain (rmMatriptase) (Catalog # 4735-SE)

• Substrate: BOC-Gln-Ala-Arg-AMC (Catalog # )

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

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

1. Dilute rmMatriptase to 0.2 ng/µL in Assay Buffer.

2. Dilute Substrate to 50 µM in Assay Buffer.

3. Load into a plate 50 µL of 0.2 ng/µL rmMatriptase, and start the reaction by adding 50 µL of 50 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 50 µM Substrate.

4. Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, in kinetic mode for 5 minutes.

5. Calculate specific activity:

     *Adjusted for Substrate Blank

     **Derived using calibration standard 7-Amino, 4-Methyl Coumarin (Sigma, Catalog # A9891).

Per Well:

• rmMatriptase: 0.010 µg

• Substrate: 25 µM

Background: Matriptase/ST14

Matriptase, a mouse type II membrane serine protease encoded by the ST14 (suppression of tumorigenicity 14) gene, is also known as epithin, and membrane-type serine protease 1/MT-SP1 (1-2). Its human ortholog MT-SP1/Matriptase (Catalog # ), which shares 81% amino acid identity with epithin, has been thought to play an important role in tumor biology and is a potential target for anti-cancer therapy (3). Matriptase has a multidomain structure containing a putative N-terminal transmembrane region, two CUB domains, four LDLRA repeats, and a C-terminal serine protease domain (1). The protease domain of epithin starts with Val615. R&D Systems recombinant mouse Matriptase is an active protease and consists of the catalytic domain (Val615 to Val855) and a short peptide (Gly596 to Arg614).

• References:

1. Cho, E. et al. (2001) J. Biol. Chem. 276:44581.

2. List, K. et al. (2006) Mol. Med. 12:1.

3. Uhland, K. (2006) Cell Mol. Life Sci. 63:2968.

• Entrez Gene IDs:

  6768 (Human); 19143 (Mouse); 114093 (Rat)

• Alternate Names:

  EC 3.4.21; Epithin; HAI; Matriptase; Membrane-type serine protease 1; MTSP1; MT-SP1EC 3.4.21.109; prostamin; PRSS14; Serine protease 14; Serine protease TADG-15; SNC19; SNC19MTSP1; ST14; suppression of tumorigenicity 14 (colon carcinoma); suppression of tumorigenicity 14 (colon carcinoma, matriptase, epithin); suppressor of tumorigenicity 14 protein; TADG15; TADG-15; TMPRSS14; tumor associated differentially expressed gene 15 protein; Tumor-associated differentially-expressed gene 15 protein