• 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     tert-butoxycarbonyl-Gln-Arg-Arg-7-amino-4-methylcoumarin (Boc-QRR-AMC). The specific activity is >8,000 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Mouse myeloma cell line, NS0-derived Gln44-Pro416 (Asp160Glu & Arg161Lys), with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  No results obtained. Gln44 inferred from enzymatic pyroglutamate treatment revealing Ser45

• Predicted Molecular Mass

  42 kDa

• SDS-PAGE

  41-47 kDa, reducing conditions

Assay Procedure

Materials

•  Activation Buffer: 0.1 M Tris, 10 mM CaCl₂, 0.15 M NaCl, 0.05% Brij-35, pH 8.0

• Assay Buffer: 50 mM Tris, pH 9.0

• Recombinant Mouse Hepsin (rmHepsin) (Catalog # 9156-SE)

• Substrate: BOC-Gln-Arg-Arg-AMC (Bachem, Catalog # I-1655), 5 mM stock in 50:50 DMSO:Methanol

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

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

1. Dilute rmHepsin to 100 μg/mL in Activation Buffer.

2. Incubate at 37 °C for 24 hours.

3. Dilute activated rmHepsin to 0.2 μg/mL in Assay Buffer.

4. Dilute Substrate to 200 μM in Assay Buffer.

5. Load into a plate 50 μL of 0.2 μg/mL rmHepsin, and start the reaction by adding 50 μL of 200 μM Substrate. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of 200 μM Substrate.

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

7. Calculate specific activity:

     *Adjusted for Substrate Blank.
     **Derived using calibration standard 7-Amino, 4-Methyl Coumain (AMC) (Sigma, Catalog # A9891).

Per Well:

• rmHepsin: 0.010 μg

• Substrate: 100 μM

Background: Hepsin

Hepsin, also known as TMPRSS1, is an approximately 50 kDa Type II membrane protein with an extracellular serine protease domain. It is primarily expressed in liver as a disulfide linked heterodimer of heavy and light chains (1, 2). Hepsin cleaves and activates Coagulation Factors VII, IX, and XII (3, 4), the pro forms of HGF, MSP, and uPA (4-8), and Prostasin/PRSS8 (9). It also cleaves EGF R and prevents receptor binding of EGF [9]. Its enzymatic activity is inhibited by HAI-1 and HAI-2 (4, 5, 8). Hepsin is overexpressed in various human tumors including prostate and breast (6, 10), and it promotes tumor progression and metastasis (11). It disrupts attachment of epithelial cells to the basement membrane by inducing the breakdown of  desmosomal structures (6). Hepsin also binds to Protein X from hepatitis B virus and to pORF3 from hepatitis E virus (12, 13). Within the ECD, mouse Hepsin shares 89% and 98% amino acid (aa) sequence identity with human and rat Hepsin, respectively (2). An alternatively spliced isoform has a 20 aa deletion in the cytoplasmic and transmembrane regions (2).

• References:

1. Tsuji, A. et al. (1991) J. Biol. Chem. 266:16948.

2. Vu, T.-K.H. et al. (1997) J. Biol. Chem. 272:31315.

3. Kazama, Y. et al. (1995) J. Biol. Chem. 270:66.

4. Herter, S. et al. (2005) Biochem. J. 390:125.

5. Kirchhofer, D. et al. (2005) FEBS Lett. 579:1945.

6. Tervonen, T.A. et al. (2016) Oncogene 35:1832.

7. Ganesan, R. et al. (2011) Mol. Cancer Res. 9:1175.

8. Moran, P. et al. (2006) J. Biol. Chem. 281:30439.

9. Chen, M. et al. (2010) Mol. Cell. Biochem. 337:259.

10. Dhanasekaran, S.M. et al. (2001) Nature 412:822.

11. Li, W. et al. (2009) Cancer Res. 69:8395.

12. Zhang, M. et al. (2015) Oncotarget 7:7780.

13. Wang, C. et al. (2014) Hepat. Mon. 14:e13902.

• Entrez Gene IDs:

  3249 (Human); 15451 (Mouse); 29135 (Rat)

• Alternate Names:

  EC 3.4.21; EC 3.4.21.106; Hepsin; HPN; serine protease hepsin catalytic chain; serine protease hepsin; TMPRSS1 ; TMPRSS1serine protease hepsin non-catalytic chain; Transmembrane protease serine 1; transmembrane protease, serine 1