• Purity

  >90%, 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, t-butoxycarbonyl-Phe-Ser-Arg-7-amino-4-methyl coumarin (Boc-FSR-AMC). The specific activity is >50,000 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Ala42-Ile418 with an N-terminal 6-His tag The protein was purified, activated and further purified.    
   

• Accession #

• N-terminal Sequence    
  Analysis

  Ile187 & His

• Structure / Form

  Active    
   

• Predicted Molecular Mass

  25 kDa & 17 kDa  

• SDS-PAGE

  27 kDa and 18 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM Tris, 0.05% (w/v) Brij-35, pH 9.5

• Recombinant Human Airway Trypsin‑like Protease/HAT (rhHAT) (Catalog # 2695-SE)

• Substrate: t-Butyloxycarbonyl Phe-Ser Arg 7-amino-4-methyl coumarin (Bachem, Catalog # I-1400), 10 mM stock in DMSO

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

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

1. Dilute rhHAT to 0.02 ng/µL in Assay Buffer.

2. Dilute Substrate to 200 µM in Assay Buffer.

3. In a plate load 50 µL of 0.02 ng/µL rhHAT, and start the reaction by adding 50 µL of 200 µM Substrate to wells. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 200 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 # A-9891).

Per Well:

• rhHAT: 0.001 µg

• Substrate: 100 µM
  

Background: Airway Trypsin-like Protease/HAT

HAT was initially purified from the sputum of patients with chronic airway diseases (1). Subsequent cloning revealed it as a member type II transmembrane serine proteases (2, 3). HAT has been shown to induce PAR-2 mediated IL-8 release in psoriasis vulgaris and increase mucin expression in airway epithelial cells (4, 5). Located in the cells of the submucosal serous glands of the bronchi and trachea, the isolated enzyme had the N-terminal sequence of ILGGTEAEEG, which corresponded to the start of the C-terminal catalytic domain (residues 187 to 418) of the deduced sequence (1, 2). The N-terminal region consisted of a short cytoplasmic tail (residues 1 to 20), a transmembrane domain (residues 21 to 41), and a SEA domain (residues 44 to 164). The ectodomain of HAT is expressed and the active enzyme is purified.

• References:

1. Yasuoka, S. et al. (1997) Am. J. Respir. Cell Mol. Biol. 16:300.

2. Yamaoka, K. et al. (1998) J. Biol. Chem. 273:11895.

3. Hooper, J.D. et al. (2001) J. Biol. Chem. 276:857.

4. Iwakiri, K. et al. (2004) J. Invest. Dermatol. 122:937.

5. Chokki, M. et al. (2004) Am. J. Respir. Cell Mol. Biol. 30:470.

• Long Name:

  Human Airway Trypsin-like Protease

• Entrez Gene IDs:

  9407 (Human)

• Alternate Names:

  airway trypsin like protease; Airway trypsin-like protease; EC 3.4.21; EC 3.4.21.-; HAT; MGC150587; MGC150588; TMPRSS11D; transmembrane protease serine 11D; transmembrane protease, serine 11D