Recombinant Mouse Tryptase gamma-1/TPSG1 Protein, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse Tryptase gamma-1/TPSG1 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ (Catalog # ES002). The specific activity is >100 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived mouse Tryptase gamma-1/TPSG1 protein
  His20-Ala275, with a C-terminal 10-His tag
  Accession # Q9QUL7
• Accession #: Q9QUL7
• N-terminal Sequence: His20
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, 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

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 28 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: 37 kDa, reducing conditions

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 opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and CaCl₂.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • 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 8.5
  • Recombinant Mouse Tryptase gamma ‑1/TPSG1 (rmTPSG1) (Catalog # 5517-SE)
  • Trypsin (Sigma, Catalog # T-1426)
  • Recombinant Human Serpin F2/ alpha ₂‑Antiplasmin (rhSerpin F2) (Catalog # 1470-PI)
  • Fluorogenic Peptide Substrate II: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(Dnp)-NH₂ (Catalog # ES002), 2 mM in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rmTPSG1 to 200 µg/mL in Activation Buffer.
  2. Dilute Trypsin to 0.164 µg/mL in Activation Buffer.
  3. Combine equal volumes of 200 µg/mL rmTPSG1 and 0.164 µg/mL Trypsin in a microfuge tube.
  4. Incubate at 37 °C for 30 min.
  5. Dilute rhSerpin F2 to 3.62 µg/mL in Assay Buffer.
  6. Add rhSerpin F2 to the reaction mixture making a ½ dilution for final concentrations of 1.81 µg/mL rhSerpin F2, 50 µg/mL rmTPSG1, and 0.041 µg/mL Trypsin.
  7. Incubate for 15 minutes at room temperature. Addition of rhSerpin F2 will stop the activity of Trypsin.
  8. Dilute rmTPSG1 to 2 ng/µL in Assay Buffer.
  9. Dilute fluorogenic peptide Substrate to 20 µM in Assay Buffer.
  10. Load into plate 50 µL of the diluted samples and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 20 µM Substrate.
  11. Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes.
  12. 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 Mca-Pro-Leu-OH (Bachem, Catalog # M-1975).

  Per Well:
  • rmTPSG1: 0.100 µg
  • Substrate: 10 µM

Notes

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

Alternate Names for Recombinant Mouse Tryptase gamma-1/TPSG1 Protein, CF

• EC 3.4.21
• EC 3.4.21.-
• gamma I
• gamma II
• lung tryptase
• mast cell protease II
• mast cell tryptase
• PRSS31skin tryptase
• Serine protease 31
• TMTpituitary tryptase
• TPSG1
• Transmembrane tryptase
• trpA
• tryptase gamma 1
• tryptase gamma I
• tryptase gamma II
• tryptase gamma
• Tryptase gamma1
• Tryptase gamma-1

Background

Tryptase gamma -1, also known as transmembrane tryptase, is a serine protease synthesized as a preproenzyme with a C-terminal transmembrane anchor (1, 2). Mast cells, mediators of inflammatory and allergic response, are found throughout the body concentrated near blood vessels in connective tissue and the mucous membranes of the respiratory and gastrointestinal tract (3). Upon activation, mast cells release granules that are enriched with neutral serine proteases including tryptases, chymase and cathepsin G (4). The recombinant mouse TPSG1 was expressed as a soluble protein terminated at residue 275, lacking the transmembrane domain, and corresponded to the proenzyme. The proenzyme can be cleaved by trypsin to form the active enzyme. The serine protease activity of trypsin-activated recombinant mouse TPSG1 can be inhibited by ecotin (Catalog # 1328-PI). Typically, >95% protease activity is inhibited by ecotin at approximately a 10:1 molar ratio.

1. Wong, G.W. et al. (1999) J. Biol. Chem. 274:30784.
2. Caughey, G.H. et al. (2000) J. Immunol. 164:6566.
3. Harris, J.L. et al. (2001) J. Biol. Chem. 276:34941.
4. Miller, H.R.P. and A.D. Pemberton (2002) Immunology 105:375.

Bioinformatics

• Uniprot:
  • Q9QUL7()