Recombinant Human Airway Trypsin-like Protease/HAT, CF

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Summary
Reactivity HuSpecies Glossary
Applications EnzAct
Format
Carrier-Free

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Recombinant Human Airway Trypsin-like Protease/HAT, CF Summary

Details of Functionality
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.
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
Ile187 & His
Structure / Form
Active
Protein/Peptide Type
Recombinant Enzymes
Gene
TMPRSS11D
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Theoretical MW
25 kDa & 17 kDa  .
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
27 kDa and 18 kDa, reducing conditions
Publications
Read Publications using
2695-SE in the following applications:

Packaging, Storage & Formulations

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 reconstitution.
Buffer
Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by silver stain
Reconstitution Instructions
Reconstitute at 100 μg/mL in sterile 25 mM Tris, 150 mM NaCl and 0.05% Brij-35, pH 7.5.
Assay Procedure
  • 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:

     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 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).

Per Well:
  • rhHAT: 0.001 µg
  • Substrate: 100 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Airway Trypsin-like Protease/HAT, CF

  • 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

Background

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.

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Publications for (3)

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Bioinformatics

Gene Symbol TMPRSS11D
Entrez
Uniprot